Regeneration and augmentation of bone using mesenchymal stem cells

ABSTRACT

Disclosed are compositions and methods for augmenting bone formation by administering isolated human mesenchymal stem cells (hMSCs) with a ceramic material or matrix or by administering hMSCs; fresh, whole marrow; or combinations thereof in a resorbable biopolymer which supports their differentiation into the osteogenic lineage. Contemplated is the delivery of (i) isolated, culture-expanded, human mesenchymal stem cells; (ii) freshly aspirated bone marrow; or (iii) their combination in a carrier material or matrix.

This application is a continuation-in-part of PCT application no.US97/06433, filed Apr. 17, 1997 (copending) which is a continuation ofU.S. provisional application Ser. No. 60/016,245, filed Apr. 19, 1996and U.S. provisional application Ser. No. 60/029,838 filed Oct. 28,1996.

Autologous, culture-expanded, bone marrow-derived MSCs have now beenshown to regenerate clinically significant bone defects. Usingtechniques for isolating and cultivating human MSCs, it should bepossible to implement therapeutic strategies based on the administrationof a patient's own cells which have been harvested by a simple iliaccrest aspiration. This method may provide an alternative to autogenousbone grafting, and will be particularly useful in clinical settings suchas ageing and osteoporosis, where the number and/or function ofendogenous MSCs have been reduced.

The repair of large segmental defects in diaphyseal bone is asignificant problem faced by orthopaedic surgeons. Although such boneloss may occur as the result of acute injury, these massive defectscommonly present secondary to congenital malformations, benign andmalignant tumors, osseous infection, and fracture non-union. The use offresh autologous bone graft material has been viewed as the historicalstandard of treatment but is associated with substantial morbidityincluding infection, malformation, pain, and loss of function (28). Thecomplications resulting from graft harvest, combined with its limitedsupply, have inspired the development of alternative strategies for therepair of clinically significant bone defects. The primary approach tothis problem has focused on the development of effective bone implantmaterials.

Three general classes of bone implants have emerged from theseinvestigational efforts, and these classes may be categorized asosteoconductive, osteoinductive, or directly osteogenic. Allograft boneis probably the best known type of osteoconductive implant. Althoughwidely used for many years, the risk of disease transmission, hostrejection, and lack of osteoinduction compromise its desirability (31).Synthetic osteoconductive implants include titanium fibermetals andceramics composed of hydroxyapatite and/or tricalcium phosphate. Thefavorably porous nature of these implants facilitate bony ingrowth, buttheir lack of osteoinductive potential limits their utility. A varietyof osteoinductive compounds have also been studied, includingdemineralized bone matrix, which is known to contain bone morphogenicproteins (BMP). Since Urist's original discovery of BMP, others havecharacterized, cloned, expressed, and implanted purified or recombinantBMPs in orthotopic sites for the repair of large bone defects(13,50,57). The success of this approach has hinged on the presence ofmesenchymal cells capable of responding to the inductive signal providedby the BMP (29). It is these mesenchymal progenitors which undergoosteogenic differentiation and are ultimately responsible forsynthesizing new bone at the surgical site.

One alternative to the osteoinductive approach is the implantation ofliving cells which are directly osteogenic. Since bone marrow has beenshown to contain a population of cells which possess osteogenicpotential, some have devised experimental therapies based on theimplantation of fresh autologous or syngeneic marrow at sites in need ofskeletal repair (15,55,56). Though sound in principle, the practicalityof obtaining enough bone marrow with the requisite number ofosteoprogenitor cells is limiting.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for directingMSCs cultivated in vitro to differentiate into specific cell lineagepathways prior to, at the time of or following, their implantation forthe therapeutic treatment of elective procedures or pathologicconditions in humans and other species. The use of both autologous andallogenic MSCs is contemplated in this invention.

The investigations reported here confirm the in vitro and in vivoosteogenic potential of MSCs; demonstrate the in vivo osteogenicpotential of MSCs when implanted at an ectopic subcutaneous site; andillustrate that purified, culture-expanded MSCs can regenerate asegmental bone defect which would otherwise result in a clinicalnon-union. These experiments compared the healing potential of MSCsdelivered in an osteoconductive or other appropriate resorbable medium.We also contemplate de novo formation of bone at the site of a desiredfusion, e.g. spinal or other joint fusions.

The invention provides a method for augmenting bone formation in anindividual in need thereof by administering isolated human mesenchymalstem cells with a matrix which supports the differentiation of such stemcells into the osteogenic lineage to an extent sufficient to generatebone formation therefrom. The matrix is preferably selected from aceramic and a resorbable biopolymer. The ceramic can be in particulateform or can be in the form of a structurally stable, three dimensionalimplant. The structurally stable, three dimensional implant can be, forexample, a cube, cylinder, block or an appropriate anatomical form. Theresorbable biopolymer is a gelatin, collagen or cellulose matrix, can bein the form of a powder or sponge, and is preferably a porcineskin-derived gelatin.

Particularly, the invention provides a method for effecting the repairor regeneration of bone defects in an animal or individual in needthereof. Such defects include, for example, segmental bone defects,non-unions, malunions or delayed unions, cysts, tumors, necroses ordevelopmental abnormalities. Other conditions requiring boneaugmentation, such as joint reconstruction, cosmetic reconstruction orbone fusion, such as spinal fusion or joint fusion, are treated in anindividual by administering, for example into the site of bone in needof augmentation, fresh whole marrow and/or isolated human mesenchymalstem cells or combinations thereof in the gelatin, cellulose or collagenbased medium to an extent sufficient to augment bone formationtherefrom. The composition can also contain one or more other componentswhich degrade, resorb or remodel at rates approximating the formation ofnew tissue.

The invention also contemplates the use of other extracellular matrixcomponents, along with the cells, so as to achieve osteoconduction orosteoinduction. In addition, by varying the ratios of the components insaid biodegradable matrices, surgical handling properties of thecell-biomatrix implants can be adjusted in a range from a dimensionallystable matrix, such as a sponge or film, to a powder.

The above method can further comprise administering to the individual atleast one bioactive factor which induces or accelerates thedifferentiation of mesenchymal stem cells into the osteogenic lineage.The MSCs can be contacted with the bioactive factor ex vivo and arepreferably contacted with the bioactive factor when the MSCs are incontact with the matrix. The bioactive factor can be, for example, asynthetic glucocorticoid, such as dexamethasone, or a bone morphogenicprotein, such as BMP-2, BMP-3, BMP-4, BMP-6 or BMP-7. The bonemorphogenic protein can be in a liquid or semi-solid carrier suitablefor intramuscular, intravenous, intramedullary or intra-articularinjection.

The invention further provides a composition for augmenting boneformation, which composition comprises a matrix selected from the groupconsisting of absorbable gelatin, cellulose and collagen in combinationwith at least one of fresh bone marrow and/or isolated mesenchymal stemcells. The composition can be used in the form of a sponge, strip,powder, gel or web. The invention also provides a method for augmentingbone formation in an individual in need thereof by administering to saidindividual a bone formation augmenting amount of the composition.

More particularly, the invention provides a method for effecting therepair of segmental bone defects, non-unions, malunions or delayedunions in an individual in need thereof by administering into the bonedefect of said person isolated human mesenchymal stem cells in a porousceramic carrier, thereby inducing the differentiation of such stem cellsinto the osteogenic lineage to an extent sufficient to generate boneformation therefrom. Preferably, the porous ceramic carrier compriseshydroxyapatite and, more preferably, the porous ceramic carrier furthercomprises β-tricalcium phosphate. The porous ceramic carrier may alsocontain one or more other biodegradable carrier components whichdegrade, resorb or remodel at rates approximating the formation of newtissue extracellular matrix or normal bone turnover.

The invention also provides for the use of other extracellular matrixcomponents, or other constituents, so as to achieve osteoconductive orosteoinductive properties similar to natural extracellular matrix. Thecomposition is an absorbable gelatin, cellulose and/or collagen-basedmatrix in combination with bone marrow and/or isolated mesenchymal stemcells. The composition can be used in the form of a sponge, strip,powder, gel, web or other physical format. The composition is, forexample, inserted in the defect and results in osteogenic healing of thedefect.

In addition, by varying the ratios of the components in saidbiodegradable matrices, surgical handling properties of thecell-biomatrix implants can be adjusted in a range from a porous ceramicblock or a moldable, putty-like consistency to a pliable gel or slurry.

More particularly, the invention comprises a rigid cell-matrix implantfor large segmental defects, spinal fusions or non-unions, gel or slurrycell-matrix implants, or infusions for stabilized fractures and othersegmental bone defects. Custom cell-matrix implants containingautologous or allogeneic MSCs can be administered using open orarthroscopic surgical techniques or percutaneous insertion, e.g. directinjection, cannulation or catheterization.

In a preferred embodiment, a composition of human mesenchymal stem cells(hMSCs) is obtained from either homogeneous, culture-expandedpreparations derived from whole-marrow (or other pre-natal or post-natalsource of autologous or allogeneic hMSCs), or from enriched orheterogenous cultures containing an effective dose of hMSCs. The key toeffective clinical outcomes using MSC therapy is to provide that numberof mesenchymal stem cells to the patient which repairs the bone or othertissue defect. This is referred to as the “Regenerative MSC Threshold”,or that concentration of MSCs necessary to achieve direct repair of thetissue defect. The Regenerative MSC Threshold will vary by: 1) type oftissue (i.e., bone, cartilage, ligament, tendon, muscle, marrow stroma,dermis and other connective tissue); 2) size or extent of tissue defect;3) formulation with pharmaceutical carrier; and 4) age of the patient.In a complete medium or chemically defined serum-free medium, isolated,culture-expanded hMSCs are capable of augmenting bone formation.

In another aspect the invention contemplates the delivery of (i)isolated, culture-expanded, human mesenchymal stem cells; (ii) freshlyaspirated bone marrow; or (iii) their combination in a carrier materialor matrix to provide for improved bone fusion area and fusion mass, whencompared to the matrix alone.

One composition of the invention is envisioned as a combination ofmaterials implanted in order to effect bone repair, osseous fusion, orbone augmentation. The components of this implanted material include, inpart, porous granular ceramic, ranging in size from 0.5 mm to 4 mm indiameter, with a preferred size ranging from 1.0 to 2.5 mm in diameter.The composition of the ceramic may range from 100% hydroxyapatite to100% tricalcium phosphate, and in the preferred form, consists of a60/40 mixture of hydroxyapatite and tricalcium phosphate. The ceramicmaterial may be uncoated, or coated with a variety of materialsincluding autologous serum, purified fibronectin, purified laminin, orother molecules that support cell adhesion. The granular ceramicmaterial can be combined with MSCs ranging in a concentration of atleast 10 thousand, more generally at least 100 thousand and morepreferably at least 1 and up to at least 3 million cells per cc. Ingeneral, the cells do not exceed 30 million cells and more generally donot exceed 10 million cells with the cells in most cases not exceeding 3million up to no more than 15 million cells per cc. It is alsoenvisioned that the cells may be in the form of fresh marrow obtainedintraoperatively, without ex vivo culture-expansion.

Bone marrow cells may be obtained from iliac crest, femora, tibiae,spine, rib or other medullary spaces. Other sources of human mesenchymalstem cells include embryonic yolk sac, placenta, umbilical cord,periosteum, fetal and adolescent skin, and blood. The cells areincubated at 37° C. with the ceramic for 0 to 5 hours, preferably 3hours. Prior to implant, the cell-loaded granules can be combined witheither fresh peripheral blood, human fibrin, fresh bone marrow, obtainedby routine aspiration, or other biological adjuvant. These finalcombinations are allowed to form a soft blood clot which helps to keepthe material together at the graft site. Implant or delivery methodsinclude open or arthroscopic surgery and direct implant by injection,e.g. syringe or cannula. Finally, these implants may be used in thepresence or absence of fixation devices, which themselves may beinternally or externally placed and secured.

The composition can also contain additional components, such asosteoinductive factors. Such osteoinductive factors include, forexample, dexamethasone, ascorbic acid-2-phosphate, β-glycerophosphateand TGF-β superfamily proteins, such as the bone morphogenic proteins(BMPs). The composition can also contain antibiotic, antimycotic,antiinflammatory, immunosuppressive and other types of therapeutic,preservative and excipient agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D. Phase contrast photomicrographs of rat MSC cultures atvarious stages of development.

FIG. 1A. A MSC colony at day seven of primary culture is composed ofuniformly spindle-shaped cells.

FIG. 1B. Passage one rat MSCs are distributed evenly across the surfaceof the dish 4 days after replating.

FIG. 1C. Rat MSCs grown in Control Medium for twenty-eight days becomeconfluent and multi-layered, but do not form mineralized nodules. APasestaining (dark gray) reveals a fraction of cells which are positive.

FIG. 1D. Rat MSC cultures grown in the presence of OsteogenicSupplements for twenty-eight days form mineralized nodules which stainblack by the von Kossa method. Cell cultures were stained by APase andvon Kossa histochemical techniques as described below (Unstained (a,b),Alkaline phosphatase histochemistry and von Kossa (c,d), all x45).

FIG. 2. Light micrograph of a representative histological section from aMSC-loaded HA/TCP implant placed ectopically in subcutaneous tissue.MSCs were loaded and the sample was implanted as described below,harvested at eight weeks, decalcified, and processed in paraffin formicroscopy. Only remnants of the HA/TCP ceramic (c) remain, while thepores of the implant are filled with bone (b), blood vessels (v), andhematopoietic elements including adipocytes (Toluidine blue-O, x70).

FIGS. 3A-3H. High resolution radiographs showing the healing of thesegmental defect at four and eight weeks with various implants. Theradiographs were obtained on a Faxitron imaging system immediatelyfollowing sacrifice. The polyethylene fixation plate is on the top ofthe bone in each radiograph. The four week radiograph is on the left,and the eight week radiograph is on the right for each group. Theradiodensity of the HA/TCP material reveals the porous nature and thecentral canal of each implant.

FIGS. 3A and 3B. Defects left empty;

FIGS. 3C and 3D. Defects fitted with HA/TCP carrier alone;

FIGS. 3E and 3F. Defects fitted with a MSC-loaded HA/TCP carrier;

FIGS. 3G and 3H. Defects fitted with a marrow-loaded HA/TCP carrier.Defects left empty following segmental gap resection undergo reactivebone formation at the cut ends of the bone, leading to a classicalnon-union in this well established model. At four weeks, the MSC-loadedsamples have begun to fill the pores of the implant material. No unionis evident in any implant type at four weeks. By eight weeks, modestunion of the host-implant interface has occurred in the carrier (d) andcarrier plus marrow groups (h), but complete integration and bonebridging is evident in the carrier plus MSC group (f). Total filling ofthe pores with bone in the MSC-loaded sample is also evident in panel F.(x1.5)

FIGS. 4A-4F. Light micrographs showing representative healing of thesegmental defect at four and eight weeks with various implant types.Intact limbs were harvested, fixed, dehydrated, cleared, embedded inpolymethylmethacrylate, cut, and ground to 100 micron thickness prior tostaining. Some animals received India ink injections to allowvisualization of the vascular tree, present here in panels B, C, D, andE as black staining. The HA/TCP material artifactually appears black inthese photomicrographs as a result of undecalcified processing. The cutedges of the host cortices are noted by arrowheads in a and b, andsimilar sections are presented in all other panels.

FIGS. 4A and 4B. Defects fitted with HA/TCP carrier alone at four andeight weeks, respectively;

FIGS. 4C and 4D. Defects fitted with a MSC-loaded HA/TCP carrier at fourand eight weeks, respectively;

FIGS. 4E and 4F. Defects fitted with a marrow-loaded HA/TCP at four andeight weeks, respectively. New bone present within the pores, or at thehost-implant interface appears blue or violet in these specimens.Importantly, only samples containing a MSC-loaded implant effectivelyheal the defect, as noted by the substantial amount of bone presentwithin the implant and at the interface with the host in panels c and d.See text for further details (Toluidine blue-O, x8).

FIGS. 5A-5B. High power light micrographs showing bone regeneration ateight weeks in segmental gaps fitted with a MSC-loaded HA/TCP implant.Panel a shows the cut edge (arrowheads) of the host cortex with new bonein direct apposition. New bone at this host-implant interface iscontiguous with bone formed in the pores of the HA/TCP carrier. Panel bshows both lamellar and woven bone (blue) filling the pores of theHA/TCP carrier. The carrier appears black in these images as anartifactual result of undecalcified specimen preparation. Blood vessels(v) which orient the secretory activity of osteoblasts are evidentwithin the pores (Toluidine blue-O, x75).

FIG. 6. Osteogenic differentiation of human MSCs in vitro. Phasecontrast photomicrographs (a, b) of human MSC cultures under growth andosteogenic conditions.

FIG. 6A. First-passage MSCs display characteristic spindle-shapedmorphology and are distributed evenly across the surface of the dishafter replating.

FIG. 6B. MSC cultures grown in the presence of OS for 16 days formmineralized nodular aggregates which stain gray for APase and black formineralized matrix (Unstained (a) x18, APase and von Kossahistochemistry (b), x45).

FIG. 6C. APase activity and calcium deposition in MSC cultures grown inControl or OS Medium on days 4, 8, 12 and 16. Samples were harvested atthe indicated days, and APase activity, cell number, and calciumdeposition were determined as described in Materials and Methods. Theresults represent the mean±SD of triplicate cultures from first passagecells. *P<0.05, †<0.005 (compared to Control).

FIG. 7. Light micrograph of a representative histological section from ahuman MSC-loaded HA/TCP implant placed ectopically in subcutaneoustissue of an athymic rat. MSCs were loaded into the ceramic, implantedas described in Materials and Methods, harvested at 12 weeks,decalcified and processed in paraffin for microscopy. Only remnants ofthe HA/TCP ceramic (c) remain, while the pores of the implant are filledwith bone (b), blood vessels (arrow) or fibrous tissue (f). Cuboidalosteoblasts are seen lining the surface of the developing bone.(Toluidine blue-O, x75).

FIG. 8. Segmental gap defect model and radiography. (a) A polyethylenefixation plate is positioned on the lateral aspect of thisrepresentative rat femur. Four bicortical screws and 2 cerclage wiresare used to secure the plate in place. An 8 mm segment of bone isremoved along with its adherent periosteum, and a ceramic implant, withor without cells, is placed into the defect site. The overlying musclesare returned to their proper anatomic position, and the skin is closedwith resorbable sutures. High resolution radiographs obtainedimmediately following sacrifice show the extent of healing of thesegmental defect at 12 weeks with the 2 implant types (b, c). Whiletotal integration of the implant at the host-ceramic interface isevident in the carrier plus MSC group (b), only modest union is observedin the cell-free implants (c). The pores of the MSC-loaded implant arefilled with bone throughout the gap, but the cell-free carrier containslittle bone and several cracks.

FIG. 9. Histologic representation of bone regeneration in segmentalfemoral defects. Immunohistochemical staining with antibody 6E2 (a)demonstrates that 4 weeks following implantation of a MSC-loaded sample,the cells within the pores of the carrier are reactive on their surface,and therefore of human origin, while cells outside the ceramic are notimmunoreactive. In phase contrast microscopy (b), the ceramic is black,and cells in the pores and surrounding the outside of the implant areevident. The ceramic material itself adsorbs fluorescent secondaryantibody and appears green (a, b, x75). Light micrographs showingrepresentative healing of a segmental defect implanted with HA/TCPcarrier alone (a), or carrier plus MSCs (c,e,f), 12 weeks afterimplantation. Limbs were harvested, fixed, dehydrated, cleared, embeddedin polymethylmethacrylate, cut, and ground to a thickness of 100 μm forstaining. The ceramic appears black in these photomicrographs as anartifact of undecalcified specimen preparation, and bone present withinthe pores or at the host-implant interface appears blue-violet. TheMSC-loaded specimen shown here was subjected to destructive mechanicaltorsion testing, and was subsequently processed for histology in twoseparate pieces. Repositioning photomicrographs of the two piecesapproximates the appearance of the femur prior to testing (c). Theactual fracture plane is denoted by the double arrows above and belowthe implant. The cut edges of the host cortices are noted by arrowheadsin c, d, and e. Only samples containing a MSC-loaded implant effectivelyheal the defect. Higher power micrographs demonstrate the substantialamount of bone present at the host-implant interface (e) and within thebody of the implant (f). (Toluidine blue-O, (c, d) x7, (e) x31, (f)x45).

FIG. 10. Radiographic images of a femoral gap in three differentcanines, 12 weeks post-implantation, with a bone-marrow-loaded gelfoamconstruct. Substantial amount of mineralized tissue is present in thedefect area in all three animals.

FIG. 11. Radiographic images of a femoral gap in three differentcanines, 16 weeks post-implantation, with a bone-marrow-loaded gelfoamconstruct. The bone defect has healed in all three cases and there is acontinuous bridge of mineralized tissue spanning the entire defect.

DETAILED DESCRIPTION OF THE INVENTION

Bone grafting procedures are widely used to treat acute fractures,fracture non-unions, bone defects, and to achieve therapeuticarthrodesis. Autogenous cancellous bone is the current “gold standard”for clinical bone grafting. Contemporary dogma attributes thiseffectiveness to three primary intrinsic properties: osteoconduction,osteogenic cells, and osteoinduction.

The marrow or isolated mesenchymal stem cells can be autologous,allogeneic or from xenogeneic sources, and can be embryonic or frompost-natal sources. Bone marrow cells may be obtained from iliac crest,femora, tibiae, spine, rib or other medullary spaces. Other sources ofhuman mesenchymal stem cells include embryonic yolk sac, placenta,umbilical cord, periosteum, fetal and adolescent skin, and blood. Inorder to obtain mesenchymal stem cells, it is necessary to isolate rarepluripotent mesenchymal stem cells from other cells in the bone marrowor other MSC source.

The present invention provides a composition for the repair of bonedefects by the rapid regeneration of healthy bone. The composition is anabsorbable gelatin, cellulose and/or collagen-based matrix incombination with bone marrow and/or isolated mesenchymal stem cells. Thecomposition can be used in the form of a sponge, strip, powder, gel, webor other physical format. The composition is, for example, inserted inthe defect and results in osteogenic healing of the defect.

The composition can also contain additional components, such asosteoinductive factors. Such osteoinductive factors include, forexample, dexamethasone, ascorbic acid-2-phosphate, β-glycerophosphateand TGF-β superfamily proteins, such as the bone morphogenic proteins(BMPs). The composition can also contain antibiotic, antimycotic,antiinflammatory, immunosuppressive and other types of therapeutic,preservative and excipient agents.

The invention also provides a method for treating a bone defect in ananimal, particularly a mammal and even more particularly a human, inneed thereof which comprises administering to the bone defect of saidanimal a bone defect-regenerative amount of the composition of theinvention.

The investigations reported here confirm the in vivo healing potentialof fresh whole marrow or MSCs delivered in the matrix alone.

The invention also contemplates the use of other extracellular matrixcomponents, along with the cells, so as to achieve osteoconductive orosteoinductive properties. In addition, by varying the ratios of thecomponents in said biodegradable matrices, surgical handling propertiesof the cell-biomatrix implants can be adjusted in a range from adimensionally stable matrix, such as a sponge or film, to a moldable,putty-like consistency to a pliable gel or slurry to a powder.

In a particularly preferred embodiment, the composition of the inventioncomprises an absorbable implant, containing whole marrow and/or isolatedMSCs for repair of segmental defects, spinal fusions or non-unions andother bone defects. Custom cell-matrix implants containing autologous,allogeneic or xenogeneic bone marrow and/or MSCs can be administeredusing open surgical techniques, arthroscopic techniques or percutaneousinjection.

Human mesenchymal stem cells (hMSCs) can be provided as eitherhomogeneous, culture-expanded preparations derived from whole-marrow (orother pre-natal or post-natal source of autologous or allogeneic hMSCs),from hMSC-enriched or heterogenous cultures containing an effective doseof at least about 10³ and preferably at least about 10⁵, preferablyabout 10⁴ or up to 10⁶, MSCs per milliliter of the composition. The keyto effective clinical outcomes, in this embodiment using MSC therapy, isto provide that number of enriched or culture-expanded mesenchymal stemcells to the patient, or about the same number in an optimized medium,which repairs the bone or other tissue defect beyond that in a volume ofwhole marrow equivalent to that of the defect. This is referred to asthe “Regenerative MSC Threshold”, or that concentration of MSCsnecessary to achieve direct repair of the tissue defect. TheRegenerative MSC Threshold will vary by: 1) type of tissue (i.e., bone,cartilage, ligament, tendon, muscle, marrow stroma, dermis and otherconnective tissue); 2) size or extent of tissue defect; 3) formulationwith pharmaceutical carrier; and 4) age of the patient.

In a preferred embodiment, the method further comprises administering atleast one bioactive factor which further induces or accelerates thedifferentiation of such mesenchymal stem cells into the osteogeniclineage. Preferably, the cells are contacted with the bioactive factorex vivo, while in the matrix, or injected into the defect site at orfollowing the implantation of the composition of the invention. It isparticularly preferred that the bioactive factor is a member of theTGF-β superfamily comprising various tissue growth factors, particularlybone morphogenic proteins, such as at least one selected from the groupconsisting of BMP-2, BMP-3, BMP-4, BMP-6 and BMP-7.

In the embodiment which uses a gelatin-based matrix, an appropriateabsorbable gelatin sponge, powder or film is cross-linked gelatin, forexample, Gelfoam® (Upjohn, Inc., Kalamazoo, Mich.) which is formed fromdenatured collagen. The absorbable gelatin-based matrix can be combinedwith the bone reparative cells and, optionally, other active ingredientsby soaking the absorbable gelatin sponge in a cell suspension of thebone marrow and/or MSC cells, where the suspension liquid can have otheractive ingredients dissolved therein. Alternately, a predeterminedamount of a cell suspension can be transferred on top of the gelatinsponge, and the cell suspension can be absorbed.

In the embodiment which uses a cellulose-based matrix, an appropriateabsorbable cellulose is regenerated oxidized cellulose sheet material,for example, Surgicel® (Johnson & Johnson, New Brunswick, N.J.) which isavailable in the form of various sized strips or Oxycel® (BectonDickinson, Franklin Lakes, N.J.) which is available in the form ofvarious sized pads, pledgets and strips. The absorbable cellulose-basedmatrix can be combined with the bone reparative cells and, optionally,other active ingredients by soaking the absorbable cellulose-basedmatrix in a cell suspension of the bone marrow and/or MSC cells, wherethe suspension liquid can have other active ingredients dissolvedtherein. Alternately, a predetermined amount of a cell suspension can betransferred on top of the cellulose-based matrix, and the cellsuspension can be absorbed.

In the embodiment which uses a collagen-based matrix, an appropriateresorbable collagen is purified bovine corium collagen, for example,Avitene® (MedChem, Woburn, Mass.) which is available in various sizes ofnonwoven web and fibrous foam, Helistat® (Marion Merrell Dow, KansasCity, Mo.) which is available in various size sponges or Hemotene®(Astra, Westborough, Mass.) which is available in powder form. Theresorbable collagen-based matrix can be combined with the bonereparative cells and, optionally, other active ingredients by soakingthe resorbable collagen-based matrix in a cell suspension of the bonemarrow and/or MSC cells, where the suspension liquid can have otheractive ingredients dissolved therein. Alternately, a predeterminedamount of a cell suspension can be transferred on top of thecollagen-based matrix, and the cell suspension can be absorbed.

The above gelatin-based, cellulose-based and collagen-based matricesmay, optionally, possess hemostatic properties.

Preferred active ingredients are those biological agents which enhancewound healing or regeneration of bone, particularly recombinantproteins. Such active ingredients are present in an amount sufficient toenhance healing of a wound, i.e., a wound healing-effective amount. Theactual amount of the active ingredient will be determined by theattending clinician and will depend on various factors such as theseverity of the wound, the condition of the patient, the age of thepatient and any collateral injuries or medical ailments possessed by thepatient. Generally, the amount of active ingredient will be in the rangeof about 1 pg/cm³ to 5 mg/cm³.

EXAMPLE 1 Rat Gap Defect Repair

Materials & Methods

Materials

Dexamethasone (Dex), sodium β-glycerophosphate (βGP), antibioticpenicillin/streptomycin, and alkaline phosphatase histochemistry kit #85were purchased from Sigma Chemical Co. (St. Louis, Mo.), DMEM-LG (DMEM)tissue culture medium from GIBCO Laboratories (Grand Island, N.Y.), andL-ascorbic acid-2-phosphate (AsAP) from Wako Chemical (Osaka, Japan).Fetal bovine serum (FBS) was purchased from GIBCO following an extensivetesting and selection protocol (35). Porous hydroxyapatite/β-tricalciumphosphate (HA/TCP) ceramic, mean pore size 200-450 μm, was generouslyprovided by Zimmer, Inc. (Warsaw, Ind.). All other routine reagents usedwere of analytical grade.

MSC Isolation and Cultivation

MSC isolation and culture expansion was performed according topreviously published methods. Briefly, male Fisher F344 rats (200-275 g)were sacrificed by pentobarbital overdose. The tibias and the femurswere recovered by dissection under sterile conditions, the metaphysealends of the bones were cut, and the marrow plugs were flushed out bypassing saline through a needle inserted into one end of the bone.Pooled marrow clots were dispersed by gentle pipetting, followed bysequential passage through a series of smaller needles yielding asingle-cell suspension. The cells were then centrifuged for ten minutesat 900×g, and resuspended in DMEM containing 10% FBS (Control Medium).Fifty million nucleated cells were plated onto petri-dishes (sixty cm²)in seven milliliters of Control Medium, and grown at 37° C. in thepresence of 5% CO₂. Non-adherent cells were removed at the time of thefirst medium change, four days post plating, and cells were routinelyfed twice weekly thereafter. These primary cultures approachedconfluence typically at thirteen days, were then released by a fiveminute exposure to 0.25% trypsin containing one millimolar EDTA, andsubcultivated at a density of 10⁴ cells/cm². Cells for implantation werederived from these first passage cultures ten days after replating, atwhich time they were approximately 85% confluent.

In Vitro Osteogenic Assays

At the end of first passage, MSCs were replated into six-well plates ata density of 10⁴ cells/cm² in Control Medium. The following day (Day 0),fresh Control Medium was provided, and the cells were grown in theabsence or presence of Osteogenic Supplements (OS) (100 nanomolar Dex,0.05 millimolar AsAP and ten millimolar β-GP) (23). Media changes wereperformed twice weekly, and at days seven, fourteen, twenty-one, andtwenty-eight, cultures were assayed for cell number, alkalinephosphatase (APase) histochemistry, and mineralized matrix productionutilizing techniques previously described (23).

Implant Preparation

HA/TCP blocks were shaped into cylinders approximately four millimeterin diameter and eight millimeter in length. A central canal roughly onemillimeter in diameter was bored through the length of the entirecylinder using an eighteen gauge hypodermic needle. Cylinders werecleaned by sonication and rinsing in distilled water, and thensterilized by 220° C. dry heat for five hours. The cylinders weresubsequently coated with human plasma fibronectin (Cal-Biochem, Irvine,Calif.) by soaking in a 100 microgram per milliliter solution forsixteen hours at 4° C. The implants were then air dried at roomtemperature overnight in a sterile biosafety cabinet, and stored at 4°C. HA/TCP cubes, measuring three millimeter per side, were similarlyprepared and coated with fibronectin as described above for use in theectopic osteogenesis assay.

HA/TCP implants, both in cube and cylinder form, were loaded with MSCsusing a modification of a technique previously described (37). Briefly,implants were placed in a suspension of MSCs (7.5×10⁶ cells/ml) in serumfree DMEM. The loading vessel was capped, and the implants weresubjected to a vacuum in three bursts of five seconds each to remove airpresent within the pores of the HA/TCP, and to facilitate fluid flowinto the pores. The loading vessels were capped loosely, placed in atissue culture incubator for two hours, and gently agitated every thirtyminutes until the time of surgery. Cell-free control cylinders weretreated identically, with the notable exception that the serum free DMEMcontained no cells. The third implant group was designed to generouslyapproximate the clinically relevant control of a fresh bone marrowaspirate. Just prior to implantation, fresh marrow cell suspensions wereobtained as previously described, centrifuged for ten minutes at 900×g,and resuspended in a volume of serum free DMEM which would coat eachcylinder with the number of bone marrow cells derived from one entirefemur, approximately fifty million (55,56). The HA/TCP implants wereloaded with this fresh marrow by rolling them in the congealed marrowsuspension.

Surgical Model and Experimental Design

The rat femoral gap model described here is a modification of one usedextensively to study long bone repair (12,21,37,50,55,56). Briefly, bothfemurs of male F344 rats (300-350 g) were exposed by an anterolateralapproach. Soft tissue and muscle was elevated while keeping theperiosteum intact along the surface of the bone. A polyethylene fixationplate (four by four by twenty-three millimeters) (Hospital for SpecialSurgery, New York, N.Y.) was secured to the anterolateral aspect of eachfemur by four threaded Kirschner-wires and two cerclage wires (Zimmer,Warsaw, Ind.). An eight millimeter transverse segment of the centraldiaphysis, along with its adherent periosteum, was removed by a rotaryosteotomy burr under saline irrigation. These stabilized segmentaldefects were either left empty, or replaced with a cell-free HA/TCPcylinder, a MSC-loaded cylinder, or a cylinder loaded with a freshmarrow cell suspension. Implants were secured by placing two 4-0 Vicryl(Ethicon, Somerville, N.J.) sutures around the ceramic and the fixationplate. The muscles were apposed, and the fascia and skin were closed ina routine layered fashion. Rats implanted with MSC-loaded cylinders alsoreceived subcutaneous implants of the MSC-loaded HA/TCP cubes tocorrelate the ectopic osteogenesis assay with orthotopic boneregeneration and the in vitro osteogenic potential of syngeneic MSCs.Rats implanted with marrow-loaded cylinders similarly receivedsubcutaneous implants of marrow-loaded cubes. The animals were allowedfull activity in their cages post-operatively. No animals experiencedfailure of fixation or other post-operative complications. At least sixlimbs were used for each of the implant groups, randomly selectedbetween left or right. Upon sacrifice at four and eight weeks, thevascular tree of some animals was perfused with India ink, and theentire femur and surrounding soft tissue was carefully dissected.Specimens were immediately evaluated radiographically, and subsequentlyprocessed for undecalcified histology.

Radiographic Analysis

The specimens were radiographed using a high resolution Faxitron Imagingsystem (Buffalo Grove, Ill.) with an exposure of thirty-five kVP forthirty seconds. The radiographs were independently evaluated by two ofthe authors who were blinded with respect to the duration and type ofimplant. Bone formation was scored on a semiquantitative scale withranges as follows: distal host-implant union (0-2); proximalhost-implant union (0-2); and implant core density (0-4). The unionscores and the core density scores were added to give a maximum possiblescore of eight for each implant. Results from both examiners wereaveraged to give final scores.

Histology and Histomorphometry

Following fixation in 10% buffered formalin, the femurs were dehydrated,cleared, and embedded in polymethylmethacrylate. Longitudinal sectionswere cut on a water-cooled Isomet saw (Buehler, Wis.), and a centralsection of each leg was ground to 100 micrometer thickness, polished,and stained with Toluidine blue-O. Leica Quantimet 500MC (Cambridge, UK)image analysis software was used to determine the area of HA/TCPimplant, bone, and soft tissue in the diaphyseal defect region of eachsection. The data were analyzed by one-way analysis of variance (ANOVA)(Sigmastat, Jandel Scientific). Further analyses were performedaccording to post hoc Student-Newman-Keuls tests. Subcutaneouslyimplanted ceramic cubes were similarly fixed in formalin, thendecalcified, dehydrated, embedded in paraffin, serially sectioned, andstained with Toluidine blue-O.

Results

MSC Cultivation and Osteogenic Differentiation in vitro

Rat MSC cultures were established from syngeneic animals and, by sevendays, formed characteristic colonies on the surface of the culture dish(FIG. 1A). Several hundred MSC colonies arose from the fifty millionnucleated cells seeded on each sixty cm² dish. On the basis of thisobservation, rat MSCs, like human MSCs (3,19), appear to be present at afrequency of approximately one in 10⁵ nucleated marrow cells. PrimaryMSC cultures subcultivated on day fourteen attached uniformly to thesurface of new dishes, and were allowed to divide for roughly ten days,or until the dishes became ˜85% confluent. Passaged cells alsodemonstrate a characteristic morphology (FIG. 1B), and uniformly divideupon the dish resulting in an even distribution of MSCs throughout theplate. Cells derived from this first passage were used for preparingimplants as described above, and an aliquot was used to confirm the invitro osteogenic potential of rat MSCs.

Seven days after replating for the osteogenic assay, both Control andOS-treated cultures were composed of spindle-shaped cells, 40-50% ofwhich were stained for APase. During the next twenty-one days, Controlcells remained fibroblastic, increased their cell surface APase, butnever underwent the morphologic changes associated with the developmentof mineralized bone nodules (FIG. 1C). By contrast, OS-treated culturesbegan to form aggregates of polygonal and cuboidal cells intenselystained for APase, and by day twenty-one, the cultures had formedcharacteristic bone-like nodules which contained von Kossa stainedmineral deposits. Further mineralization of these nodules through daytwenty-eight (FIG. 1D) was accompanied by a decrease in APase staining,especially within the internodular regions.

MSC-Mediated Osteogenesis in Ectopic HA/TCP Implants

All MSC-loaded HA/TCP cubes implanted in the host rats had ampleevidence of osteogenesis by four weeks. At the eight week time point, asubstantial amount of bone, and occasionally cartilage, was presentwithin the pores of the cubes. A representative section from aMSC-loaded cube harvested eight weeks following implantation is shown inFIG. 2. The unstained granular areas reflect the former regions ofceramic material which have been removed during the decalcification stepof specimen preparation. As seen in the photomicrograph, bone formationoccurs within the pores of the cubes, and is associated with vascularelements which penetrate the implant. Such angiogenesis is obligatory tonew bone formation since the secretory activity of osteoblasts is anoriented phenomenon guided by vasculature. Both woven and lamellar bonecan be seen depending on the duration of implantation, and the preciseregion examined. Most of the pores are filled with bone and smallislands of hematopoietic elements, with the remainder being filled witha loose connective tissue. In contrast to these MSC-loaded samples,cubes loaded with fresh marrow contained negligible osseous tissue atfour weeks, and only slightly more even at eight weeks. As previouslydemonstrated (19), cubes implanted without MSCs or marrow contained nobone, but were filled with fibrous tissue and blood vessels.

Radiographic Evaluation

High resolution Faxitron radiographs provided sufficient clarity anddetail to discern subtle changes occurring within the implant and thesurrounding host bone. FIG. 3 shows representative radiographs of thefemurs from each of the groups recovered at four and eight weekspost-implantation. As demonstrated in these radiographs, the fixationremained intact in all the samples and there were no fractures in any ofthe femurs. In animals whose femoral defects were left empty, reactivebone formation at the transversely cut edges of the host femur wasobserved at four weeks (FIG. 3A). By eight weeks, slightly more bone waspresent within the gap, however, most of this bone appeared to formalong the edge of the fixation plate which was in contact with theperiosteum (FIG. 3B). Every specimen which was left empty resulted inthe formation of a radiographic non-union. Some limbs, irrespective ofthe group, also contained an eccentric spicule of bone which was usuallyon the outside of the defect opposite the fixation plate.

When the HA/TCP cylinder was implanted, negligible reactive boneformation occurred at the cut edges of the femur. Due to the mineralcontent of the implant material (HA/TCP), one can appreciate thestructural details of the implant itself upon radiographic evaluation.The details of the central canal and pores are clearly visible in thefour week radiographs (FIG. 3C), and serve to provide an importantbaseline for comparison to the other radiographic images. Blurring ofthe pore margins can be appreciated by eight weeks (FIG. 3D) in thesecell-free implants. Importantly, the lack of union between the implantand the host is manifested as a clear zone of radiolucency between theimplant itself and the cut edges of the femur in all animals at fourweeks. In contrast to the four week carrier alone, animals whichreceived MSC-loaded HA/TCP cylinders demonstrated substantial new boneformation within the pores of the implant by four weeks (FIG. 3E).Increasing radiodensity, and obliteration of the apparent porestructure, was used as an indication of new bone formation within thecore of the implant. Although integration of the implant, or union, wasnot observed by four weeks, the subsequent formation of a radiodensebone bridge between the implant and the host completely masked theinterface. By eight weeks, the MSC-loaded implant was contiguous andcompletely integrated with the normal host bone (FIG. 3F). HA/TCPimplants which were loaded with fresh marrow did not appear to produceradiodense bone within the pores at either time point, although modestintegration with the cut ends of the host bone was evident by eightweeks (FIGS. 3G and 3H).

The average of the radiographic scores at each time point for eachimplant group is provided in Table 1.

TABLE 1 Average of Radiographic Scores for Each Implant Group Four WeeksEight Weeks C C only C + MSCs C + M only C + MSCs C + M Proximal 1.5 0.80.2 1.2 1.3 1.0 Union Distal Union 0.5 1.4 0.7 1.2 2.0 1.6 Core Density0.7 2.0 0.3 0.6 3.7 0.7 Total Score 2.7 4.2 1.2 3.0 7.0* 3.3 Table 1.Average of radiographic scores for each implant group at each timepoint. C = carrier, M = marrow. Radiographs were evaluated and scored bytwo independent observers blinded to the identity of each implant. Unionwas scored both proximally and distally on a scale of 0-2. Core densitywas scored on a scale of 0-4. n = 3 for each group at each time point.The maximum possible total score is 8. One- # way analysis of varianceat the two different time points, with cell loading (none, MSCs, andmarrow) as the independent variable showed significant differencebetween groups at 8 weeks (F = 10.9, p = 0.01) but were notsignificantly different at four weeks. * = significantly greater (p <0.05) than other groups at the corresponding time point (according topost hoc Student-Newman-Keuls tests).

In the case of the defects filled with the HA/TCP carrier alone, the lowscores indicate the absence of any radiodense material within the pores,and minimal union of the implant with the host bone. Loading the HA/TCPimplant with fresh marrow did not result in an improvement in thehealing of the defect, and the low scores reflect the similarity of thisgroup to that of the carrier alone. However, loading the HA/TCP carrierwith MSCs produces a vigorous osteogenic response. Even at four weeks,pore filling was observed and is reflected in the considerably higherscores of these implants. Interestingly, even in this case thehost-implant union was modest compared to controls. By the eight weektime point, the pores of the implant were filled with new bone and thehost-implant union was well established.

Histologic and Histomorphometric Evaluation

Histologic evaluation of the samples confirmed the observations made byradiography. In the empty defects, reactive bone formation appeared toemanate from the cut ends of the host cortices and endosteum. Even ateight weeks there was no bridging across the defect, and a fibrousnon-union had formed at the center of the segmental gap.Photomicrographs of representative sections of the implant groupsrecovered at four and eight weeks are shown in FIG. 4. In defects fittedwith the HA/TCP carrier alone, the pores of the implant were filled withfibrous tissue (FIG. 4A) and were well vascularized as determined byIndia Ink injection. No bone could be seen within the pores of theimplant and there was limited integration with the host. Even at eightweeks, most of the pores were devoid of any bone despite significantvascularization evident in this photomicrograph (FIG. 4B). A smallamount of new bone was present at the host-implant interfaces, and atone end of this representative implant, host-derived endosteal boneappears to be advancing into the medullary canal of the implant. Boneformation in samples loaded with fresh marrow was very similar to thatof the HA/TCP carrier alone (FIGS. 4E and F). However, a modest amountof new bone could be seen within the pores of the implant at eightweeks, correlating with the results of the ectopic implants. Union ofthese implants was similar to that observed with cell-free implants;reactive bone formation slightly penetrated the pores at the ends of theimplant.

In contrast to the sparse osteogenesis resulting from the addition offresh marrow to the HA/TCP, most of the pores of the implants loadedwith MSCs contained considerable new bone by four weeks (FIG. 4C).Again, there was still a clear demarcation between the cut edges of thehost bone and the ends of the implant. At eight weeks nearly every porewas filled with new bone, except in some discrete areas where loading ofthe MSCs may have been suboptimal. Interestingly, substantial new boneformation occurred at the interface between the host and the implant,leading to a continuous span of bone across the defect (FIG. 4D).Furthermore, a periosteal callus was also present in samples loaded withMSCs (FIG. 4D), but not in other implant types. The bone formed withinthe pores and at the ends of these implants represents de novo boneformation, is highly cellular, and is presented in higher magnificationphotomicrographs in FIG. 5. New woven and lamellar bone can be seen inintimate contact with the cut edge of the host cortex at eight weeks(FIG. 5A). Importantly, this region of union is directly contiguous withbone formed throughout the pores of implant. In regions deeper withinthe HA/TCP, filling of the pores with new bone is evident, as is theassociation of vasculature which orients the secretory activity of thedifferentiating osteoblasts (FIG. 5B).

The results qualitatively described above are mirrored in thehistomorphometric data presented in Table 2.

TABLE 2 Carrier alone Carrier + MSCs Carrier + Marrow 2.3 ± 1.5 19.3 ±3.7* 2.9 ± 1.7 10.4 ± 2.4 43.3 ± 7.7* 17.2 ± 6.0 Table 2. Bone fill inHA/TCP implants as a percentage of available space. Histomorphometricmeasurements were obtained on the bone formed within the confines of thesegmental resection, excluding the implant material itself and themedullary canal. The values are reported as means of three samples alongwith standard deviations from the mean. One-way analysis of variance atthe two different time points, with cell loading (none, MSCs, andmarrow) as the independent variable showed # significant differencebetween MSC-loaded samples at both 4 weeks (F = 43.3, p < 0.001) and 8weeks (F = 26.2, p < 0.002). * = significantly greater (p < 0.01) thanother groups at the corresponding time points (according to post hocStudent-Newman-Keuls tests). No difference was observed between marrowand carrier alone at either time point (p > 0.1).

The cell-free HA/TCP implants had a bone fraction of only 2.3% and 10.4%at four and eight weeks, respectively. Importantly, this fraction ofbone at eight weeks correlates with previously published results (126).These fractions primarily represent the bone ingrowth from the cut endsof the host cortices. The marrow-loaded HA/TCP cylinders did exhibitmodest osteogenesis within the body of the implant and consequently hada slightly higher value of 17.2% at eight weeks. Importantly, by fourweeks, the MSC-loaded samples exceeded the eight week value for theother two groups. The 19.3% bone fill at this four week time point ismost likely attributable to MSC-mediated osteogenesis. The average bonefraction within the implant increased over time, reaching 43% by eightweeks. One-way ANOVA performed on the data along with theStudent-Newman-Keuls tests showed that at both four and eight weeks, theMSC treatment was significantly better than the carrier alone or themarrow-loaded carrier (p<0.01). No significant difference betweencarrier alone and marrow-loaded implants was detected. The volumefraction of the HA/TCP carrier remained constant, and served as aninternal control for the histomorphometry system. Even though the emptydefects had 34% bone fill by eight weeks, there was no bridging acrossthe defect, and thus would be classified as a clinical non-union.

Discussion

In the present study, we have demonstrated that purified,culture-expanded syngeneic progenitor cells are capable of healing aclinically significant bone defect in a well established animal model.These progenitor cells are referred to as mesenchymal stem cells sincethey give rise not only to bone (1,19,23), but to cartilage (24,35,53),muscle (48,54), tendon (6), and a stromal tissue which supportshematopoietic differentiation (38). While the osteogenic potential ofboth animal and human MSCs has been proven via subcutaneous implants inectopic assays, rigorous and quantitative studies establishing theability of culture-expanded MSCs to regenerate large segmental bonedefects have not been reported to our knowledge. The combination of MSCswith a porous HA/TCP implant material are shown in the present study tobe an effective strategy for healing large segmental bone defects. Thecurrent investigation further substantiates that compared to freshmarrow, MSCs produce significantly more bone when placed in either anectopic or an orthotopic site. With these results as a foundation, wemay begin to refine our approach to cell therapies for the regenerationof skeletal defects.

To further characterize the cells used in this study, we cultured themin the presence and absence of a medium which induces osteogenicdifferentiation in vitro. As has been reported in numerous otherlaboratories (32,39,47,52), these rat marrow-derived cells develop alongthe osteogenic lineage in response to dexamethasone, eventually formingmineralized nodules of bone-like tissue on the surface of the dish. Suchdifferentiation is evident in our photomicrographs (FIG. 1), and servesto document that the cells used in these implants indeed possess theability to form bone, one of the inherent properties of MSCs.Furthermore, the bone and cartilage formed in cubes implantedsubcutaneously not only confirms the osteochondral potential of theMSCs, but acts as an internal control to verify that every host rat wascapable of providing an environment which could support osteogenesiswithin these combined cell:matrix implants. Additional experimentsdocumenting the multilineage potential of these cells were not includedas part of the current study because previous publications have focusedon describing such potential in greater detail (34,35,48,54). Theisolation and selection procedures for rat MSCs are similar to thoseused for human MSCs (19,35), and result in the formation ofcharacteristic primary colonies illustrated in FIG. 1A. These cells aremitotically expanded to yield a morphologically homogeneous populationwhich divides uniformly across the dish. Both human and rat MSCs havebeen shown to possess multilineage potential, and the details of invitro osteogenic differentiation of human MSCs has recently beenreported (10,23). Conditions for the isolation and culture expansion ofhuman MSCs without lineage progression have been optimized (3,19,35),and the development of a serum free medium for human MSC growth has beencompleted (58).

The radiographic findings in this study establish a precedent forobtaining non-invasive evidence of bone regeneration in animals, orhumans, which receive MSCs in an orthotopic location. Given the porousnature of the HA/TCP implants, new bone which forms within theinterstices of the material is readily apparent radiographically by fourweeks, in spite of the inherent radiodensity of the HA/TCP material. Theprogressive increase in radiodensity evident by eight weeks correlateswell with the histological observations of processed limbs.Interestingly, despite the presence of new bone within the core ofimplants by four weeks, integration at the host-implant interface wasnot observed until eight weeks. The mean radiographic scores for thethree implant groups document a significant (p<0.05) difference betweenMSCs and either marrow-loaded and ceramic implants at eight weeks, whileno significant difference was observed between marrow-loaded and ceramicimplants at either time point.

The histologic studies demonstrate appositional bone growth on thesurface of the HA/TCP throughout the core of the implant, consistentwith previous observations of osteogenesis in ectopic implants loadedwith MSCs (19). The bone which is formed at four and eight weeks inMSC-loaded samples is woven in many areas, but lamellar bone can also beappreciated (FIGS. 5A and 5B). It is critical to note that in theprocess of regenerating this osseous defect, bone formation occurs by adirect conversion of mesenchymal cells into osteoblasts rather than byan endochondral sequence. As regeneration of the bone at the defect sitecontinues, the pores of the ceramic are filled with significantly morebone, which is laid down upon the walls of the implant or existing bone,and oriented by the invading vasculature. These blood vessels,visualized by India inking of animals immediately prior to sacrifice,also provide a portal for the entry and establishment of new marrowislands which contain hematopoietic elements, as well as host-derivedMSCs. The process of bone remodeling ensues, and eventually the donorbone is replaced by host bone. At the edge of the defect, integration ofthe implant is achieved with direct continuity between the cut edge ofthe host cortex and the new bone formed upon the surface of the implant(FIG. 5A). Since only minimal host-implant union occurs in rats providedwith either marrow-loaded or cell-free ceramics, the advancedintegration observed in MSC-loaded ceramics likely reflects the combinedcontributions of implanted MSCs and host-derived cells. The lack ofearly union in all samples was surprising in light of the fact thatdefects which were left empty underwent a substantial amount of reactivebone formation at the cut edges of the cortices. It is possible that thepresence of an implant in the defect site inhibits migration and/orprolapse of the surrounding loose mesenchyme which contributes to thereactive bone formation in the empty defects. Furthermore, micromotionof the implanted cylinders would likely hinder stable union at theinterface.

The ability of MSCs to regenerate a large segmental defect in thisexperimental model compares favorably with other investigations testingimplants such as demineralized bone matrix, bone marrow, purified orrecombinant BMPs, allograft, ceramics, and fibermetals(12,29,37,50,55,56). While the use of recombinant BMP has receivedconsiderable attention, the precise mechanism of action has onlyrecently been appreciated. These powerful inductive molecules act onundifferentiated mesenchymal cells to initiate the endochondral cascade,ultimately resulting in the formation of bone. Studies ofundifferentiated rat marrow stromal cells confirm that BMP-2 acts todirectly stimulate osteoblast development, and that this stimulation isenhanced by the addition of dexamethasone (32). Others have shown thatbone formation occurs in an orthotopic site when fresh marrow alone isadded, but the rate and extent of healing is a function of the amount ofmarrow and the number of osteoprogenitor cells residing therein (15,55).An important set of experiments by Takagi and Urist demonstrate that theaddition of BMP is not effective at healing segmental defects whenaccess to the medullary canal and the marrow stroma is prevented, thusindicating an absolute requirement for the cellular constituents ofmarrow in BMP-mediated bone repair. These results were bolstered bystudies indicating that the implantation of fresh marrow along with BMPin a rat segmental gap model is more effective than either componentimplanted alone (29). One may conclude from all of the above thatmarrow-derived mesenchymal progenitors, or MSCs, are the target forendogenous osteoinductive molecules, such as BMPs, which are releasedduring normal bone healing. It therefore follows that one must have anadequate supply of MSCs in order to respond to the normal (orexogenously supplied) signals of bone repair, or healing will be effete.

The histomorphometric data generated in this study provides a basis forcomparison to other investigations. When fresh marrow from one femurequivalent is loaded on an HA/TCP implant, no significant difference inbone formation is observed when compared to implants which receive nocells. This is true for both time points in our study, and likelyreflects an inadequate number of MSCs in the volume of marrow applied.Had we loaded the implants with considerably more marrow, we and otherswould predict that greater healing of the bone defect would haveoccurred (29,55). Nevertheless, an appropriate clinically relevantcontrol is generously approximated by applying the total cell populationobtained from one long bone since removing all the marrow from multiplelong bones for the repair of a focal defect is contradictory to soundclinical judgment. Perhaps most importantly, MSCs produced a bone fillof 19.3% and 43.2%, respectively, at four and eight weeks. When purifiedBMP was applied to an identical carrier in the same experimental model,the bone fill was 21% at four weeks, and only 22% by eight weeks (50).These BMP-coated HA/TCP implants did not achieve a bone fill of 43percent until 16 weeks following implantation. While similar amounts ofbone resulted from both implant types at four weeks, MSCs produce twiceas much bone as BMP by the eight week time point. In this formulation,it took BMP sixteen weeks to form the same amount of bone which MSCsproduce in only eight weeks. On this basis, it appears that MSCs offer aconsiderable advantage to the use of BMP alone, although somecombination of BMP and MSCs could provide an even faster, more vigorousbone repair as discussed above.

Since the number of progenitor cells present at the site of repair is acritical factor, it is obligatory to estimate how the MSC-loadedimplants compare with marrow-loaded implants in this regard. The numberof nucleated marrow cells which were placed on an implant wasapproximately fifty million; the same number harvested from one longbone. Another fifty million cells were used to initiate the MSC culturewhich eventually provided cells for one implant. From these fiftymillion cells, roughly 500 MSC colonies develop, and these cells aremitotically expanded to three million by the end of first passage. Thisrepresents a 6,000-fold increase in MSC number due to approximatelytwelve population doublings. Using the current technique to load thesetype of implants, it appears that only about 150,000 cells becomeadherent following incubation with the MSC suspension. Nevertheless, thelocal administration of 150,000 purified MSCs would increase the numberof progenitor cells 300 times over the number normally present in fiftymillion unfractionated marrow cells. On the basis of these calculations,the advantage which this technique offers over other bone regenerationstrategies is direct delivery of the cellular machinery required forbone formation. This approach would have an extraordinary advantage insettings where the number of endogenous progenitor cells is reduced,such as that which occurs in ageing, osteoporosis, or a variety of otherpathologic conditions (10,28,36,47,51,52). Other investigators havepursued this logic by attempting to deliver more progenitor cells simplyby concentrating the marrow, by crude fractionation and removal of redblood cells, or by cultivating the stromal cells in vitro (37,41,55).Now that techniques and conditions have been established which supportthe expansion of purified human MSCs in culture as much as one billionfold without a loss in osteogenic potential (3), analogous clinicalprotocols for regenerating human bone defects are not far away. It willbe possible to further expedite the healing process by directing theseculture-expanded MSCs ex vivo to enter the osteogenic lineage prior toimplantation, thus decreasing the in situ interval between implantationand their biosynthetic activity as osteoblasts. Additional efforts areunderway to develop cell delivery vehicles which will provide moreflexibility to the surgeon, including materials which can be shaped tofit any type of defect. By combining a pharmacologic stimulus, such asBMP, with an even better delivery vehicle, we will be able to offerpatients therapeutic options which have never before been available.

EXAMPLE 2 Large Segmental Canine Femoral Defects are Healed withAutologous Mesenchymal Stem Cell Therapy

This study demonstrates that culture-expanded, autologous mesenchymalstem cells can regenerate clinically significant bone defects in a largeanimal model.

Recently, the ability of syngeneic bone marrow-derived mesenchymal stemcells (MSCs) to repair large segmental defects in rodents wasestablished (25). These MSCs may be isolated from marrow or periosteum,expanded in number ex vivo, and delivered back to the host in anappropriate carrier vehicle. Studies in rats demonstrated that theamount of bone formed 8 weeks following implantation of MSCs was twicethat resulting from BMP delivered in the same carrier (25,50). In orderto demonstrate clinical feasibility of this technology, our objectivewas to regenerate segmental bone defects in a large animal amenable tostringent biomechanical testing. To achieve this goal, we developed acanine femoral gap model to compare radiographic, histologic, andbiomechanical data following implantation of an MSC-loaded carrier,carrier alone, and cancellous autograft bone.

Materials and Methods

MSC Cultivation and Manipulation

A 15 cc bone marrow aspirate was obtained from the iliac crest of eachanimal, according to an IACUC-approved protocol, and shipped on ice byovernight courier to the cell culture facilities. Isolation of canineMSCs was achieved by centrifuging whole marrow aspirates over a Percollcushion, using procedures analogous to those developed for human MSCisolation (19). Tissue culture flasks (185 cm²) were seeded with 10⁷nucleated cells isolated from the cushion, and cultured with DMEMcontaining 10% fetal calf serum from a selected lot (35). Cells werepassaged at 8×10³ cells/cm², and transported back to the veterinaryhospital where they were maintained until the time of implantation.Cell-loaded implants were prepared by incubating fibronectin-coatedporous hydroxyapatite-tricalcium phosphate (HA/TCP) cylinders (Zimmer,Inc.) in a 7.5×10⁶ cells/ml suspension of MSCs for 3 hr at 37° C. Theinterval between marrow harvest and implantation was 16 days. An aliquotof cells from each preparation was also cultured under osteoinductiveconditions to quantify aspects of osteoblastic differentiation.

Canine Femoral Gap Model

A unilateral segmental femoral defect model was developed for this studyfollowing IACUC approval. Under general anesthesia, thirty-sixskeletally mature female purpose-bred hounds (20 kg) underwent resectionof a 21 mm long osteoperiosteal segment from their mid-diaphysis. A 4.5mm Synthes® 8-hole lengthening plate was contoured to the lateral aspectof the bone, and secured with bicortical screws. The defect was filledwith one of three materials; 1) a cell-free HA/TCP cylinder, 2) anMSC-loaded HA/TCP cylinder, or 3) cancellous bone harvested from theiliac crest. HA/TCP implants were secured by placing two sutures aroundthe implant and the plate. Animals received peri-operative antibiotics,and analgesics were administered for three days post-operatively.

Radiographic and Histologic Analyses

Standard radiographic images were obtained at pre-op, immediatelypost-op, and at 4 week intervals until termination of the study. Allsamples contained a radiodensity step wedge to provide a basis forcomparing changes over time, and between dogs. Upon sacrifice, specimenswere subjected to high resolution Faxitron radiography, and subsequentlyprocessed for biomechanical evaluation. Following torsion testing,undecalcified longitudinal sections will be processed for quantitativehistomorphometry.

Biomechanical Testing

Sixteen weeks after implantation, animals were sacrificed for torsiontesting of femurs. The fixation plate, screws, and adherent soft tissuewere removed, and the metaphyses of the bones were embedded. Thespecimens were externally rotated in a custom torsion test apparatus,failure load and stiffness recorded, and the data analyzed by one wayANOVA according to post hoc Student-Newman-Keuls tests.

Results

All animals tolerated the surgical procedure well, with no incidence ofinfection, implant rejection, or failure of fixation. Two modes ofrepair were apparent in the MSC-loaded samples; first, considerablecallus formation occurred at both host-implant interfaces; and second, asubstantial collar of bone surrounding the implant itself developed.Cell-free implants did not possess either of these features. Autograftsamples underwent a traditional consolidation sequence, with themajority of bone laid down in the medial aspect of the gap defect.MSC-loaded samples not only became fully integrated at the host implantinterface, but the periosteal collar extended proximally and distallybeyond the cut edges of the gap. Furthermore, the diameter of new boneat the mid-diaphysis was greater in MSC-loaded implants than eitherautograft samples or intact limbs. in vitro analyses of the osteogenicpotential MSCs from each animal demonstrate the development of alkalinephosphatase-positive cells which deposit significant mineralizedextracellular matrix.

TABLE 3 Histomorphometric and Histologic Features of Bone RegenerationSixteen Weeks Following Implantation of Ceramic Cylinders (C) or CeramicCylinders plus Autologous Mesenchymal Stem Cells (M) Average ThicknessBone Soft of Peri-implant Ceramic Area Tissue Callus (Medial InterfaceDog # Area (%) (%) Area (%) Surface) (mm) Union C1 34.0 7.6 92.4 0.00 1C2 33.6 35.4 64.6 0.00 4 C3 45.3 7.3 92.6 0.00 — C4 33.7 23.5 76.5 0.004 C5 35.9 47.0 53.0 0.00 4 C6 28.1 23.1 76.9 0.00 4 Mean 35.1 24.0 76.00.00 3.4 M1 40.2 41.9 58.1 0.33 4 M2 38.2 38.5 61.5 3.17 4 M3 33.8 42.657.4 2.50 4 M4 39.2 37.0 63.0 0.67 4 M5 28.0 30.5 69.5 2.00 4 M6 32.648.7 51.3 0.67 4 Mean 35.3 39.9* 60.1* 1.56* 4.0 *P < 0.05 compared toControl implants.

In the case of the MSC-loaded samples, in addition to the considerableamount of bone in the confines of the ceramic block, there was also afairly large mineralized periosteal callus. Also, the marrow space wasreestablished within the defect. Whereas in the cell-free HA/TCPcylinders, most of the bone present was in the endosteal space with somepenetration into the implant.

Torsional testing of the samples (n=6 per group) showed that theMSC-loaded samples were almost twice as long as the cell free examples,but were only a third as strong as autograft controls.

Discussion

The present study demonstrates that MSCs from a large animal may beculture-expanded, and implanted for the successful repair of largediaphyseal bone defects. Radiographic and histologic evidence indicatesthat not only do the MSCs form bone within and around the implantdirectly, but their presence elicits a response in the host periosteumto form additional bone. The mechanism of this is currently not known,but is consistent with our observation that MSCs undergoing osteogenicdifferentiation secrete a paracrine factor(s) which is osteoinductive(22). The conspicuous lack of callus formation and periosteal reactionin the cell-free implants was an unexpected finding. In addition toestablishing a new standardized model for large animal bone repair, thisstudy illustrates the feasibility of translating autologous stem celltherapy from the laboratory into the clinic.

EXAMPLE 3 in vivo Bone Formation Using Human Mesenchymal Stem Cells

Although rat MSCs have been shown to synthesize structurally competentbone in an orthotopic site (25), human MSCs have only been shown to formbone in vitro (2,23) and in an ectopic implantation site inimmunodeficient mice. Since fracture healing and bone repair depend onthe ability to amass enough cells at the defect site to form a repairblastema, one therapeutic strategy is to directly administer theprecursor cells to the site in need of repair. This approach isparticularly attractive for patients who have fractures which aredifficult to heal, or patients who have a decline in their MSCrepository as a result of age (28,47), osteoporosis (51), or othermetabolic derangement. With this in mind, the goal of the current studywas to show that purified, culture-expanded human MSCs are capable ofregenerating bone at the site of a clinically significant defect.

Materials and Methods

Human MSC Cultivation and Manipulation

Isolation and culture-expansion of human MSCs from a bone marrowaspirate obtained from a normal volunteer after informed consent wasconducted as previously described (17,19). Following initial plating inDulbecco's Modified Eagle's Medium (Sigma) containing 10% fetal bovineserum (BioCell) from a selected lot (35), non-adherent cells wereremoved on day 3 at the time of the first medium change, and freshmedium was replaced twice weekly thereafter. Adherent MSCs representapproximately 1 in 10⁵ nucleated cells originally plated. When culturedishes became near-confluent, cells were detached and seriallysubcultured.

In Vitro Osteogenic Assays

Human MSCs were replated into six-well dishes at a density of 3×10³cells/cm². The following day (Day 0), fresh medium was provided, and thecells were grown in the absence or presence of Osteogenic Supplements(OS) (2,23). Media changes were performed twice weekly, and at days 4,8, 12 and 16, cultures were assayed for cell number, alkalinephosphatase (APase) biochemistry and histochemistry, and mineralizedmatrix production utilizing techniques previously described (23).

Implant Preparation

Porous hydroxyapatite/β-tricalcium phosphate (HA/TCP) ceramic blocks,mean pore size 200-450 μm (Zimmer, Inc., Warsaw, Ind.), were shaped intocylinders approximately 4 mm in diameter and 8 mm in length with a 1 mmcentral canal, or cut into cubes 3 mm per side. MSC-loaded implants wereprepared by incubating human fibronectin-coated HA/TCP cubes andcylinders in a 7.5×10⁶ cell/ml suspension of first passage MSCs for 2 hrat 37° C. as previously described (25). Cell-free control cylinders wereprepared identically.

Athymic Rat Femoral Gap Model

The femoral gap surgical model employed here has been used extensivelyin euthymic rats to study long bone repair (25). Briefly, both femurs ofHarlan Nude (Hsd:Rh-rnu) rats (325 g) were exposed by an anterolateralapproach. A polyethylene fixation plate was attached to each femur byfour Kirschner wires, and an 8 mm transverse segment of the centraldiaphysis, along with its adherent periosteum, was removed by using arotary osteotomy burr under saline irrigation. Each animal then receiveda cell-free HA/TCP cylinder in one femoral defect, an identical cylinderloaded with human MSCs in the contralateral defect, and a subcutaneousimplant of a MSC-loaded HA/TCP cube along the dorsum.

Radiography

Immediately after sacrifice at each time point, all specimens wereradiographed in a lateral position using a high resolution FaxitronImaging system with an exposure of 35 kVP for 30 sec.

Quantitative Histomorphometry and Immunochemistry

Upon sacrifice at 4, 8, and 12 weeks, a minimum of 3 specimens of eachtype were processed for undecalcified histology following radiography.Longitudinal sections were cut, stained with Toluidine blue-O, andquantitative assessment of bone formation was performed using LeicaQuantimet 500MC image analysis software as previously described (25).The data were analyzed by Student's t-test. Subcutaneously implantedsamples were fixed in formalin, decalcified, embedded in paraffin,serially sectioned, and similarly stained. Limbs from one animal at eachtime point were also prepared for immunostaining by monoclonal antibody6E2, which distinguishes human cells from rat cells (19). Undecalcifiedcryosections were incubated with 6E2 supernatant, or an irrelevantprimary monoclonal antibody control (SB-1), followed by FITC-conjugatedgoat anti-mouse IgG secondary antibody (GIBCO) diluted 1:500 inphosphate-buffered saline.

Biomechanical Testing

Twelve weeks after implantation, 7 experimental animals and 6 unoperatedcontrol animals were sacrificed for torsion testing of femurs aspreviously described (81). The fixation plate and adherent soft tissuewere removed, and the metaphyses of the bones were embedded. Thespecimens were externally rotated in a custom torsion test apparatus,failure load and stiffness recorded, and the data analyzed by one wayANOVA with post hoc Student-Newman-Keuls tests.

Results

MSC Cultivation and Osteogenic Differentiation in vitro

Human MSC cultures were established and, by 7 days, formedcharacteristic colonies on the surface of the culture dish. Primarycolonies which were subcultivated on day 14 attached uniformly to thesurface of new dishes, and were allowed to divide for another 7 daysuntil they became ˜85% confluent. Passaged cells demonstrated theircharacteristic spindle-shaped morphology (FIG. 6A), and uniformlydivided resulting in an even distribution of MSCs throughout the plate.Cells derived from this first passage were used for preparing implantsas previously described, and an aliquot was used to confirm theirosteogenic potential in vitro.

As described in previous studies (2,3,23), MSCs cultured with OSunderwent a dramatic change in cellular morphology from that ofspindle-shaped to cuboidal, which was accompanied by an increase inAPase activity and production of an extracellular matrix rich in bonehydroxyapatite (FIG. 6B). A significant increase in APase activity wasobserved after 4 days of OS treatment with maximal activity occurring onday 8, followed by a decline through day 16 (FIG. 6C). This latedecrease in APase activity of OS cultures correlates with increasingmineral deposition and terminal differentiation of cells intoosteocytes. While no calcium deposition was detected either by Von Kossastaining or the sensitive colorimetric quantitative calcium assay inControl cultures, FIG. 6C illustrates that MSCs grown with OS depositeda significant amount of calcium by days 12 (60±5.1 μg/dish) and 16(98±5.0 μg/dish).

MSC-Mediated Osteogenesis in Ectopic HA/TCP Implants

Human MSC-loaded HA/TCP cubes implanted in the subcutaneous space ofathymic rats displayed evidence of osteogenesis by 4 weeks, butconsiderably more bone was present within the pores at 8 and 12 weeks. Arepresentative section from a MSC-loaded cube harvested 12 weeksfollowing implantation is shown in FIG. 7. Bone formation occurs withinthe pores of the cubes, and is associated with vascular elements whichpenetrate the implant. Such angiogenesis is obligatory to new boneformation since the secretory activity of osteoblasts is an orientedphenomenon guided by vasculature (8). As previously demonstrated (19),cubes implanted without MSCs never contained bone but were filled withfibrous tissue and blood vessels only.

Osteotomy Model and Radiography

FIG. 8A illustrates the segmental defect model used in this study. Thepolyethylene fixation plate on top of the femur provides stabilityfollowing creation of the 8 mm diaphyseal defect. No animals experiencedfailure of fixation or other post-operative complications throughout thecourse of study. Previous studies have established that femoral defectsthat are not implanted with a bioactive material give rise to a fibrousnon-union devoid of bone (25). High resolution Faxitron radiographsprovided sufficient clarity and detail to discern subtle changesoccurring within the implant and the surrounding host bone.Representative radiographs of the femurs from the 2 groups recovered 12weeks post-implantation demonstrate substantially more bone in animalswhich received MSC-loaded HA/TCP cylinders (FIG. 8B) versus cell-freecylinders (FIG. 8C). Increasing radiodensity, and obliteration of theapparent pore structure, was used as an indication of new bone formationwithin the core of the implant. Although integration of the implant, orunion, was not generally observed by 4 weeks, the subsequent formationof a radiodense bone bridge between the implant and the host at 8 weekscompletely masked the interface. By 8 weeks, the MSC-loaded implantcontained considerable bone within the pores and was integrated with thehost bone at the ends of the implant. At 12 weeks, union was completeand additional bone was evident in the pores. Callus formation along thefixation plate was observed in some samples, as was an occasionaleccentric spicule of bone usually present along the medial aspect of thefemur. Some specimens, both with and without cells, contained crackswithin the core of the implant.

Immunocytochemical Evaluation

Immunocytochemical staining with antibody 6E2 demonstrates that, at 4weeks, virtually all the cells within the pores of the implant werereactive on their surface and were, therefore, of human origin (FIG.9A). Along the immediate periphery of the implant, the host rat cellswere intermingled with the human donor cells, but as the distance awayfrom the surface of the implant increased, the representation of donorcells precipitously declined. The presence of these peripheral cellswhich are not immunostained also serves as a negative control for thisestablished antibody. The ceramic material itself, which appears blackin the phase contrast micrograph (FIG. 9B), displays a high level ofbackground fluorescence. The exquisite sensitivity of the 6E2antigen:antibody interaction necessitated that we use unfixed frozensections which, unfortunately, limited our ability to process thesecalcified tissue specimens for immunostaining. While we were able toobtain satisfactory cryosections of 4 week samples (shown here), we wereunable to prepare sections from later samples which containedsubstantially more bone.

Histologic Evaluation

Analysis of the Toluidine blue-O-stained samples confirmed theobservations made by radiography. Photomicrographs of representativesections of the implant groups recovered at 12 weeks are shown in FIG.9. Most of the pores of the implants loaded with MSCs containedsubstantial new bone by 8 weeks, and this process of bone regenerationcontinued through the 12 week assessment period (FIG. 9C). At 8 weeksnearly all pores contained new bone, except in some discrete areas whereloading of the MSCs may have been compromised. Evaluation of limbsfollowing biomechanical testing indicates that fractures were of atransverse or spiral nature, and were generally propagated through acentral region of the implant containing cartilage or a modest amount ofbone, as seen in FIG. 9C. During the regenerative process, substantialnew bone formation occurred at the interface between the host and theimplant, leading to a continuous span of bone across the defect. Newwoven and lamellar bone can be seen in intimate contact with the cutedge of the host cortex at 12 weeks (FIG. 9E), and this region of unionis directly contiguous with bone formed throughout the pores of implant.In regions deeper within the HA/TCP (FIG. 9F), filling of the pores withnew bone and vasculature is evident.

In defects fitted with the HA/TCP carrier alone, the pores of theimplant were predominantly filled with fibrous tissue even at 12 weeks(FIG. 9D). Many samples had evidence of modest integration at thehost-implant interfaces, and at one end of this representative implant(FIG. 9D), host-derived endosteal bone appears to be advancing into themedullary canal of the carrier as a result of osteoconduction. None ofthe cell-free ceramic carriers contained bone throughout the pores ofthe implant.

Histomorphometric Evaluation

The results qualitatively described above are mirrored in thehistomorphometric data presented in Table 4.

TABLE 4 Bone Fill in HA/TCP Implants as a Percentage of Available SpaceFour Weeks Eight Weeks Twelve Weeks Carrier Alone 1.89 ± 1.00 11.47 ±7.08 29.51 ± 8.93 Carrier plus MSCs 1.95 ± 1.92 26.46 ± 3.60* 46.61 ±14.83* Table 4. Longitudinal sections through the segmental defect ofathymic rats implanted with ceramic carriers, with and without humanMSCs, were evaluated histomorphometrically for bone content. The resultsrepresent the mean ± SD of 3 experimental limbs of each group at 4 and 8weeks, and 8 limbs of each group at 12 weeks. *P < 0.05 compared to thecarrier alone at each time point.

Bone present in the cell-free HA/TCP implants primarily represents thebony ingrowth from the cut ends of the host cortices. At 4 weeks andbeyond, the MSC-loaded samples contained significantly more bone thanthe cell-free group, and the average bone fraction within the implantincreased over time, reaching 26.5% and 46.6% by the 8 and 12 week timepoints, respectively. This increased bone fraction at 8 weeks is2.3-fold higher than that measured in cell-free implants at the sametime, and by 12 weeks, is over 23-fold higher than that observed ineither condition at 4 weeks. The volume fraction of the HA/TCP carrierremained constant, and served as an internal control forhistomorphometry.

Mechanical Testing

Twelve experimental and 11 intact femora from age and weight-matchedcontrol animals were tested in torsion 12 weeks after implantation. Twoexperimental limbs were not tested because they were extremely fragile.Gross inspection of the healed defects revealed a distal varus rotationdeformation in most specimens. Table 5 summarizes the mechanical testingresults in terms of torsional strength, stiffness, and total energyabsorbed.

TABLE 5 Mechanical Properties of Rat Femora 12 Weeks after ImplantationIntact Control Carrier Alone Carrier + MSCs Strength(N mm) 409 ± 71 74 ±63 159 ± 37 Stiffness(N mm/deg) 39 ± 5.5 6.6 ± 4.2 16.2 ± 4.0 Energy(Nmm × deg) 2.6 ± 0.7 0.6 ± 0.4 1.3 ± 0.8 Table 5. Mechanical testing dataon rat femur samples from unoperated age matched controls (IntactControl), or animals whose segmental defects were implanted with theHA/TCP carrier alone (Carrier alone) or the MSC-loaded HA/TCP (Carrier +MSCs). These results represent the mean ± SD of 6 limbs from eachexperimental implant group, and 11 limbs from control animals. Twelveweeks after implantation, each specimen was harvested, the ends of the #bone were embedded, and the samples were tested in external rotation at6 degrees/second along the longitudinal axis until failure. One-wayANOVA on each of the parameters showed a significant difference betweenthe groups at P < 0.0001. Furthermore, each of the groups weresignificantly different from the other for strength and stiffness (P <0.05), as determined by post hoc Student-Newman-Keuls tests.

These results demonstrate a 115%, 145% and 112% increase in strength,stiffness and torsional energy absorbed, respectively, in MSC-loadedsamples compared to cell-free carrier samples. All three groups werefound to be statistically different from each other in failure torqueand stiffness.

Discussion

The results presented here demonstrate that purified, culture-expandedhuman MSCs are capable of healing a clinically significant bone defectin a well-established model for bone repair. While the osteogenicpotential of human MSCs has been proven by neo-osteogenesis insubcutaneous implants (19), as well as in studies of isolated MSCs invitro (2,23), this is the first demonstration that human MSCs can formbone at an orthotopic site in need of repair. The combination of MSCswith a porous HA/TCP carrier possesses regenerative potential which ishistomorphometrically and biomechanically superior to the carrier alone.This investigation paves the way for the clinical application ofautologous MSC-therapy for the treatment of orthopedic defects in man.

The progressive increase in radiodensity of the healing bone at 8 weeksparallels the histological observations of processed limbs.Immunocytochemistry proves that the cells associated with the ceramic at4 weeks are of human origin, and that the cells surrounding the implantare from the host. At 8 weeks and beyond, bone is laid down by the donorMSCs and eventually resorbed and replaced by bone derived from hostcells through the normal remodeling sequence (7). It is important tonote that in the process of regenerating this osseous defect, boneformation occurs by a direct conversion of mesenchymal cells intoosteoblasts rather than by an endochondral cascade. This observation isconsistent with previous studies of osteogenesis in implants loaded withanimal or human MSCs (18,27,25,68). As the regenerative processcontinues, the pores of the ceramic are filled with an increasing amountof bone, which is laid down upon the walls of the implant or existingbone, and oriented by the invading vasculature that provides a portalfor the entry and establishment of new marrow islands containinghematopoietic elements and host-derived MSCs.

The rate of bone regeneration is lower than that observed in euthymicrats implanted with syngeneic MSCs (25), suggesting thatimmunocompromised rats are not the ideal hosts to assess thebone-forming potential of human MSCs. This may be due in part to thexenogeneic nature of the implant and the increased natural killer cellactivity, which may be a compensatory mechanism for the animal to copewith its deficient T-cell-mediated immunity (49). Nevertheless, asignificantly higher amount of bone was formed in the defect whichreceived MSCs compared to those limbs receiving the carrier only. Theextent of host-implant union was greater in the MSC-loaded implants,which likely reflects the combined contributions of implanted MSCs andhost-derived cells.

The ability of human MSCs to regenerate bone in this experimental modelcompares favorably with other investigations testing implants such asdemineralized bone matrix, bone marrow, purified or recombinant bonemorphogenic proteins (BMP), allograft, ceramics, fibermetals andgene-activated matrices. In addition to forming a substantial amount ofhistologically normal bone, the biomechanical data demonstrate thattorsional strength and stiffness at 12 weeks were ˜40% that of intactcontrol limbs, which is more than twice that observed with the cell-freecarrier, and also twice that achieved in a similar study of bone repairusing fresh autograft in a primate long bone defect model (9).

Recently, growth factors such as recombinant human BMP have beenimplanted in experimental bone defect models in an effort to stimulatebone repair (33,9). Although recombinant BMPs are capable of inducingthe endochondral cascade in ectopic implants (57), their ability toreproducibly direct bone formation at orthotopic sites has been hamperedby the problems associated with the design and selection of anappropriate carrier. In contrast to the mechanical data showingsignificant bone regeneration in a MSC-loaded ceramic, BMP delivered inthe same HA/TCP carrier did not increase implant strength over thecarrier alone (50). The brittle nature of this ceramic, combined withits slow resorption and complex porous structure, may explain why evenin the presence of significant bone formation mechanical strengthremains less than intact limbs. In addition, stress shielding of the newbone, as a result of the load-bearing fixation plate, also restricts thestrength of the healing defect. We believe, as has been previouslysuggested, that the use of an osteosupportive HA/TCP cylinder may not bethe ideal matrix for replacement of diaphyseal defects. Efforts atdesigning the optimal biomatrix carrier for the delivery of MSCs is anactive area of investigation.

Implantation of culture-expanded autologous MSCs offers the advantage ofdirectly delivering the cellular machinery responsible for synthesizingnew bone, and circumventing the otherwise slow steps leading to bonerepair. Even in patients with a reduced ability to regenerate connectivetissue, presumably due to a low titer of endogenous MSCs (28,51,55,1),these rare MSCs may be isolated and culture-expanded over onebillion-fold without a loss in their osteogenic potential (3), thusrestoring or enhancing a patient's ability to heal tissue defects. Thestudies presented here suggest that MSC-based cell therapies will beuseful for the reconstruction of a variety of tissue defects in man.

EXAMPLE 4 Effect of Coating on the Osteogenic Response of MSC-LoadedHA/TCP Cubes

This experiment was performed in an attempt to establish that uncoatedHA/TCP cubes are equivalent to fibronectin- or autologous serum-coatedHA/TCP cubes in supporting MSC-mediated osteogenesis.

Materials & Methods

Standard HA/TCP cubes coated with either fibronectin, 1% autologousserum, 10% autologous serum or those left uncoated, were loaded withMSCs and implanted subcutaneously into athymic mice. The cubes wereretrieved six weeks post-implantation and inspected for the level ofosteogenesis by decalcified histological methods. The experiments weredone with multiple human and canine donors, and were performed induplicate mice.

Results & Conclusion

MSC-loaded cubes from all treatment groups showed a significant amountof bone formation at six weeks. The coating of HA/TCP cubes with eitherfibronectin or serum had no effect on the level of MSC-mediatedosteogenesis within the cube. As expected, the cell-free control HA/TCPcubes did not have osteogenesis. Based on the above results, we concludethat uncoated HA/TCP is a viable carrier for the delivery of MSCs toeffect bone repair/augmentation.

EXAMPLE 5 Bone Defect Repair Using Bone Marrow in an AbsorbableGelatin-Containing Sponge

The objectives of this study were to demonstrate efficacy of bone marrowand/or mesenchymal stem cells (MSCs) in healing clinically significantbone defects in an established animal model.

Materials & Methods

In the study, Fisher 344 rats (Charles River Laboratories, Wilmington,Mass.) of approximately 325 grams in weight were used. A bilateralfemoral gap 8 mm in length was created in each femur. This length isapproximately towards the diameter of the mid-diaphysis of the femur. Aninternal fixation plate was applied with four Kirschner wires. Thegroups for comparison were separately treated with one of the following:

(1) Gelfoam® sterile sponge (Upjohn—Kalamazoo, Mich.);

(2) Peripheral blood clot plus marrow derived from four bones;

(3) Gelfoam® sponge containing marrow derived from four bones;

(4) Gelfoam® sponge plus varying amounts of marrow from one bone down toone-half of one bone in the presence of fresh peripheral blood toprovide clot.

In this animal system, fresh marrow from four bones yields approximately150 million cells while fresh marrow from one-half of one bone yieldsapproximately 20 million nucleated cells. Each group consisted of aminimum of three animals, all of which were sacrificed six weekspost-operatively to obtain the desired end-points. Some animals receivedhigh-resolution Faxitron radiographs at an intermediate point threeweeks after implantation. At the six-week time point when all animalswere sacrificed, the limbs were removed, radiographed, and prepared forundecalcified histological evaluation.

Handling properties of the Gelfoam® sponge, in combination with freshmarrow in the presence or absence of fresh peripheral blood clot, wasdesirable and nearly equivalent.

Results

Evaluation of radiographs following sacrifice of the animals at 6 weeksrevealed no bone in the defect region of those animals implanted witheither Gelfoam® sponge alone or those animals implanted with freshmarrow and a peripheral clot. Minimal endosteal spiking of new bone atthe cut edges of the defect was observed, as is the case with thehistorical control of no implant alone. By contrast, those animalsreceiving Gelfoam® sponge plus marrow from four bones or one bone, inthe absence or presence of peripheral clot, demonstrated a robustosteogenic healing response in the region of the implant. Those animalsimplanted with Gelfoam® sponge and marrow from one-half of one bone inthe presence of peripheral clot respectively demonstrated only modestamounts of bone formation. Finally, those animals implanted withGelfoam® sponge and marrow from one-half of one bone in the absence offresh peripheral clot demonstrated no bone in the defect region.Histologic analysis of all of the specimens confirms the observationsmade based on high-resolution radiographs. The formation of neocorticesin samples of Gelfoam® sponge loaded with marrow cells was impressive.Histologic evaluation also indicates that no residual Gelfoam® materialwas retained at the site of the implant six weeks following surgery.Samples of Gelfoam® sponge loaded with marrow from one bone demonstratedislands of developing hemaetopoietic elements in the medullary canal.Host-implant interfaces appear to be intact.

EXAMPLE 6 Bone Defect Repair in a Canine Model

A 21 mm segmental defect was created in three adult female dogs, andstabilized with a stainless steel orthopaedic plate and screws. Thedefects were filled with autologous marrow-loaded Gelfoam® sponge. Toconstruct the marrow-loaded sponge, first a piece of Gelfoam® (size 100)sponge (28 mm×21 mm) was hydrated in PBS. Then the sponge was blotted toremove the PBS, folded over and placed into a 10 cc Terumo syringe fromwhich the tip had been cut off. Within five minutes of blotting, 4 cc offreshly aspirated bone marrow from the iliac crest was added to thesponge and allowed to soak into the sponge. After an incubation time of30-45 minutes to allow clotting of the marrow in the sample, thecylindrical sample was extruded out of the syringe and placed into thedefect. The size of the syringe, the dimensions of the sponge, and themarrow volume used was chosen such that the construct aproximated thesize and shape of the resected segment.

The dogs were radiographed post-operatively and subsequently every fourweeks until sacrifice at sixteen weeks. The femurs containing theimplants were harvested at sacrifice and undecalcified histology wasperformed on the samples.

There were no implant failures or other complications with this study.The radiographs were evaluated for healing of the gap and gradedaccording to previously published ordinal scale ranging from 0-4 (Table6). By 12 weeks, a substantial amount of mineralized tissue was presentin the defect area in all three animals (FIG. 10) and by 16 weeks therewas a continuous bridge of mineralized tissue spanning the entire defect(FIG. 11). The histologic data confirmed the radiographic conclusionsand in all cases the defect was found to have healed by the 16 weekstime point. This was in contrast to defects that had been left empty,which were subsequently found to have only a minimal amount of bone inthe defect, and this bone was limited to the regions at the cut edges ofthe defect. See Johnson et al., J. Orthop. res., 14:351-369, 1996.

TABLE 6 Dog Post-Op 4 weeks 8 weeks 12 weeks 16 weeks 3C1 0 1 2 4 4 3C20 1 2 4 4 3C3 0 1 2 4 4

In summary, significant osteogenic response of syngeneic marrow in eachof the recipient dogs which were implanted with Gelfoam® spongeindicates the suitability of this cell and matrix combinationimplantation for the repair of significant bone defects.

Cited Literature

1. Bruder, S. P.; Fink, D. J.; and Caplan, A. I.: Mesenchymal stem cellsin bone development, bone repair, and skeletal regeneration therapy. J.Cell. Biochem. 56:283-294, 1994.

2. Bruder, S. P.; Eames, B. F.; and Haynesworth, S. E.: Osteogenicinduction of purified human mesenchymal stem cells in vitro:Quantitative assessment of the osteoblastic phenotype. Trans. Ortho.Res. Soc. 20:464, 1995

3. Bruder, S. P.; Jaiswal, N.; Haynesworth, S. E.: Growth kinetics,self-renewal and the osteogenic potential of purified human mesenchymalstem cells during extensive subcultivation and followingcryopreservation, (1997) J. Cell Biochem. 64(2):278-294.

4. Bruder, S. P., Lawrence, E. G., and Haynesworth, S. E. (1995) Trans.Ortho. Res. Soc. 20, 8.

5. Bucholz, R. W., Carlton, A., and Holmes, R. E. (1987) Orthop. Clin.North Am. 18, 323-334.

6. Caplan, A. I.; Fink, D. J.; Goto, T.; Linton, A. E.; Young, R. G.;Wakitani, S.; Goldberg, V. M.; and Haynesworth, S. E.: Mesenchymal stemcells and tissue repair. In The Anterior Cruciate Ligament: Current andFuture Concepts. D. W. Jackson, ed. Raven Press, Ltd., New York.405-417, 1993.

7. Caplan, A. I., and Bruder, S. P. (1997) in Textbook of TissueEngineering, eds. Lanza, R., Langer, R., and Chick, W. (R.G. LandesCompany, Georgetown), pp. 603-618.

8. Caplan, A. I. and Pechak, D. (1987) in Bone and Mineral Research/5,ed. Peck, W. A. (Elsevier, New York), pp. 117-183.

9. Cook, S. D., Wolfe, M. W., Salkeld, S. L., and Rueger, D. C. (1995)J. Bone Joint Surg. 77-A, 734-750.

10. Egrise, D.; Martin, D.; Vienne, A.; Neve, P.; and Schoutens, A.: Thenumber of fibroblastic colonies formed from bone marrow is decreased andthe in vitro proliferation rate of trabecular bone cells increased inaged rats. Bone 13:355-361, 1992.

11. Fang, J., Zhu, Y-Y., Smiley, E., Bonadio, J., Rouleau, J. P.,Goldstein, S. A., McCauley, L. K., Davidson, B. L., and Roessler, B. J.(1996) Proc. Natl. Acad. Sci. USA 93, 5753-5758.

12. Feighan, J. E.; Davy, D.; Prewett, A.; and Stevenson, S: Inductionof bone by a demineralized bone matrix gel: a study in a rat femoraldefect model. J. Orthop. Res. 13:881-891, 1995.

13. Gerhart, T. N.; Kirker-Head, K.; Kriz, M. J.; Holtrop, M. E.;Hennig, G. E.; Hipp, J.; Schelling, S. H.; and Wang, E.: Healingsegmental femoral defects in sheep using recombinant human bonemorphogenic protein. Clin. Orthop. Rel. Res. 293:317-326, 1993.

14. Grande, D. A., Southerland, S. S., Manji, R., Pate, D. W., Schwartz,S. E., and Lucas, P. A. (1995) Tissue Engin. 1(4), 345-353.

15. Grundel, R. E.; Chapman, M. W.; Yee, T.; and Moore, D. C.:Autogeneic bone marrow and porous biphasic calcium phosphate ceramic forsegmental bone defects in the canine ulna. Clin. Orthop. Rel. Res.266:244-258, 1991.

16. Haynesworth, S. E., Baber, M. A, and Caplan, A. I. (1995) Trans.Ortho. Res. Soc. 20, 7.

17. Haynesworth, S. E., Baber, M. A, and Caplan, A. I. (1996) J. CellPhysiol. 166(3), 585-592.

18. Haynesworth S E, Baber M A, and Caplan A l.: Cell surface antigenson human marrow-derived mesenchymal cells are detected by monocionalantibodies. Bone 13:69-80, 1992.

19. Haynesworth, S. E.; Goshima, J.; Goldberg, V. M.; and Caplan, A. I.:Characterization of cells with osteogenic potential from human marrow.Bone. 13:81-88, 1992.

20. Holocek, J.; Lennon, D. L., Haynesworth, S. E.; Marshak, D. R.; andCaplan, A. I: Unpublished data.

21. Hunt, T. R.; Schwappach, J. R.; and Anderson, H. C.: Healing of asegmental defect in the rat femur with use of an extract from a culturedhuman osteosarcoma cell-line (Saos-2). J. Bone Joint Surg. 78(1):41-48,1996.

22. Jaiswal, N. and Bruder, S. P. Trans. O.R.S.: 524, 1997.

23. Jaiswal, N.; Haynesworth, S. E.; Caplan, A. I.; and Bruder, S. P.:Osteogenic differentiation of purified, culture-expanded humanmesenchymal stem cells in vitro, (1997) J. Cell Biochem. 64(2):295-312.

24. Johnstone, B.; Yoo, J. U.; Barry, F. P.: in vitro chondrogenesis ofbone marrow-derived mesenchymal cells. Trans. Ortho. Res. Soc. 21: 65,1996.

25. Kadiyala, S., Jaiswal, N., and Bruder, S. P. (1997) Tissue. Engin.3, Volume 3, Number 2, 173-185: Culture-expanded, bone marrow-derivedmesenchymal stem cells can regenerate a critical-sized segmental bonedefect.

26. Kadiyala, S., Kraus, K. H., and Bruder, S. P. (1996) Trans. Tissue.Engin. Soc. 1, 20.

27. Kadiyala, S., Young, R. G., Thiede, M. A., and Bruder, S. P. (1997)Cell Transplant. 6, Volume 6, Number 2, 125-134: Culture-expanded caninemesenchymal stem cells possess osteochondrogenic potential in vivo andin vitro.

28. Kahn, A.; Gibbons, R.; Perkins, S.; and Gazit, D.: Age-related boneloss: A hypothesis and initial assessment in mice. Clin. Orthop. Rel.Res. 313:69-75, 1995.

29. Lane, J. M.; Yasko, A.; Tomin, E.; Bostrom, M.; Rosen, V.; andWozney, J.: Orthopaedic application of BMP-2 in fracture healing. InFirst International Conference on Bone Morphogenic Proteins, Baltimore,Md., Jun. 8-11 (abstract), 1994.

30. Laurie, S. W. S.; Kaban, L. B.; Mulliken, J. B.; and Murray, J. E.:Donor-site morbidity after harvesting rib and iliac bone. Plast.Reconstr. Surg. 73(6):933-938, 1984.

31. Leads from the MMWR. Transmission of HIV through bonetransplantation: Case report and publich health recommendations. JAMA.260:2487-2488, 1988.

32. LeBoy, P. S.; Beresford, J.; Devlin, C.; and Owen, M.: Dexamethasoneinduction of osteoblast mRNAs in rat marrow stromal cell cultures. J.Cell Physiol. 146:370-378, 1991.

33. Lee, S. C., Shea, M., Battle, M. A., Kozitza, K., Ron, E., Turek,T., Schaub, R. G., and Hayes, W. C. (1994) J. Biomed. Mater. Res. 28,1149-1156.

34. Lennon, D. P.; Haynesworth, S. E.; Young, R. G.; Dennis, J. E.; andCaplan, A. I.: A chemically defined medium supports in vitroproliferation and maintains the osteochondral potential of ratmarrow-derived mesenchymal stem cells. Exp. Cell Res. 219:211-222, 1995.

35. Lennon, D. P.; Haynesworth, S. E.; Bruder, S. P.; Jaiswal, N.; andCaplan, A. I.: Human and animal mesenchymal progenitor cells from bonemarrow: Identification of serum for optimal selection and proliferation.in vitro Cell. Dev. Biol., 32(10):602-611, 1996.

36. Liang, C. T.; Barnes, J.; Seedor, J. G; Quartuccio, H. A.; Bolander,M.; Jeffrey, J. J.; and Rodan, G. A.: Impaired bone activity in agedrats: Alterations at the cellular and molecular levels. Bone.13:435-441, 1992.

37. Liebergall, M.; Young, R. G.; Ozawa, N.; Reese, J.; Davy, D. T.;Goldberg, V. M.; and Caplan, A. I.: The effects of cellular manipulationand TGF-β in a composite bone graft. In: Bone Formation and Repair.Brighton, C., Friedlander, G., and Lane, J. (eds), American Academy ofOrthopaedic Surgeons, Rosemont, Ill., 367-378, 1994.

38. Majumdar, M. K.; Haynesworth, S. E.; Thiede, M. A.; Marshak, D. R.;Caplan, A. I.; and Gerson, S. L.: Culture-expanded human mesenchymalstem cells (MSCs) express cytokines and support hematopoiesis in vitro.Blood 86(10):494a (1995).

39. Malaval, L.; Modrowski, D.; Ashwani, G.; and Aubin, J. E.: Cellularexpression of bone-related proteins during in vitro osteogenesis in ratbone marrow stromal cell cultures. J. Cell Physiol. 158:555-572, 1994.

40. Mosca, J. D., Majumdar, M. K., Hardy, W. B., Pittenger, M. F., andThiede, M. A. (1997) Blood 88(10), 186a.

41. Niedzwiedzki, T.; Dabrowski, Z.; Miszta, H.; and Pawlikowski, M.:Bone healing after bone marrow stromal cell transplantation to the bonedefect. Biomaterials 14:115-121, 1993.

42. Ou Y, Piedmonte M R, and Medendrop S V.: Latent variable models forclustered ordinal data. Submitted to Biometrics.

43. Owen, M.; Lineage of osteogenic cells and their relationship to thestromal system. In Bone and Mineral/3. W. A. Peck, ed. Elsevier,Amsterdam, 1-25, 1985.

44. Owen, M.: Marrow stromal stem cells. J. Cell Sci. Suppl. 10:63-76,1988.

45. Pereira, R. F.; Halford, K. W.; O'Hara, M. D.; Leeper, D. B.;Sokolov, B. P.; Pollard, M. D.; Bagasra, O.; and Prockop, D. J.: Cultureadherent cells from marrow can serve as long-lasting precursor cells forbone, cartilage, and lung in irradiated mice. Proc. Natl. Acad. Sci.USA. 92:4857-4861, 1988.

46. Pittenger, M. F., Mackay, A. M., and Beck, S. C. (1996) Mol. Biol.Cell. 7, 582a.

47. Quarto, R.; Thomas, D.; and Liang, T.: Bone progenitor cell deficitsand the age-associated decline in bone repair capacity. Calcif. TissueInt. 56:123-129, 1995.

48. Saito, T.; Dennis, J. E.; Lennon, D. P.; Young, R. G.; and Caplan,A. I.: Myogenic expression of mesenchymal stem cells within myotubes ofmdx mice in vitro and in vivo. Tissue. Engin. 1(4):327-343, 1995.

49. Schuurman, H.-J., Hougen, H. P., and van Loveren, H. (1992) ILARJournal. 34(1-2), 3-12.

50. Stevenson, S.; Cunningham, N.; Toth, J.; Davy, D.; and Reddi, A. H.:The effect of osteogenin (a bone morphogenic protein) on the formationof bone in orthotopic segmental defects in rats. J. Bone Joint Surg.76(11):1676-1687. 1994.

51. Tabuchi, C.; Simmon, D. J.; Fausto, A.; Russell, J.; Binderman, I.;and Avioli, L.: Bone deficit in ovariectomized rats. J. Clin. Invest.78:637-642, 1986.

52. Tsuji, T.; Hughhes, F. J.; McCulloch, C. A.; and Melchher, A. H.:Effect of donor age on osteogenic cells of rat bone marrow in vitro.Mech. Ageing Dev. 51:121-132, 1990.

53. Wakitani, S.; Gotto, T.; Pineda, S. J.; Young, R. G.; Mansour, J.M.; Caplan, A. I.; and Goldberg, V. M.: Mesenchymal cell-based repair oflarge, full-thickness defects of articular cartilage J. Bone Joint Surg.76A:579-592, 1994.

54. Wakitani, S.; Saito, T.; and Caplan, A. I.: Myogenic cells derivedfrom rat bone marrow mesenchymal stem cells exposed to 5-azacytidine.Muscle & Nerve 18:1417-1426, 1995.

55. Werntz, J. R.; Lane, J. M.; Burstein, A. H.; Justin, R.; Klein, R.;and Tomin, E.: Qualitative and quantitative analysis of orthotopic boneregeneration by marrow. J. Orthop. Res. 14:85-93, 1996.

56. Wolff, D.; Goldberg, V. M.; and Stevenson, S.: Histomorphometricanalysis of the repair of a segmental diaphyseal defect with ceramic andtitanium fibermetal implants: Effects of bone marrow. J. Orthop. Res.12:439-446, 1994.

57. Wozney, J. M.; Rosen, V.; Celeste, A. J.; Mitsock, L. M.; Whitters,M. J.; Kriz, R. W.; Hewick, R. M.; and Wang, E. A.: Novel regulators ofbone formation: Molecular clones and activities. Science. 242:1528-1534,1988.

58. Young, R. G., Butler, D. L., Weber, W., Gordon, S. L., and Fink, D.J. (1997) Trans. Ortho. Res. Soc. 22, 249.

What is claimed is:
 1. A method of augmenting bone formation in anindividual in need thereof by administering to said individual isolatedhuman mesenchymal stem cells with a resorbable biopolymer selected fromthe group consisting of a gelatin, collagen, and cellulose, wherein saidresorbable biopolymer supports the differentiation of such stem cellsinto the osteogenic lineage to an extent sufficient to generate boneformation therefrom.
 2. A composition for augmenting bone formation,said composition comprising a resorbable biopolymer selected from thegroup consisting of gelatin, cellulose, and collagen, in combinationwith isolated mesenchymal stem cells.
 3. A composition for augmentingbone formation, said composition comprising a resorbable biopolymerselected from the group consisting of gelatin, cellulose, and collagen,wherein said resorbable biopolymer is a sponge, strip, film, gel, orweb, or a structurally stable, three dimensional implant, in combinationwith isolated mesenchymal stem cells.
 4. A method for augmenting boneformation in an individual in need thereof which comprises administeringto said individual a composition comprising a resorbable biopolymerselected from the group consisting of gelatin, cellulose, and collagenin combination with isolated mesenchymal stem cells.
 5. The method ofclaim 1 wherein the medium is a powder, sponge, strip, film, gel or webor a structurally stable, three dimensional implant in the form of acube, cylinder or block or in the shape of an anatomical form.
 6. Themethod of claim 1 wherein the resorbable biopolymer is a porcineskin-derived gelatin.
 7. The method of claim 1 which further comprisesadministering to said individual at least one bioactive factor whichinduces or accelerates the differentiation of such mesenchymal stemcells into the osteogenic lineage.
 8. The method of claim 7 wherein thecells are contacted with the bioactive factor ex vivo.
 9. The method ofclaim 8 wherein the cells are contacted with the bioactive factor whenin contact with the matrix which supports the differentiation of suchstem cells into the osteogenic lineage to an extent sufficient togenerate bone formation therefrom.
 10. The method of claim 7 wherein thebioactive factor is a synthetic glucocorticoid.
 11. The method of claim10 wherein the synthetic glucocorticoid is dexamethasone.
 12. The methodof claim 7 wherein the bioactive factor is a bone morphogenic protein.13. The method of claim 12 wherein the bone morphogenic protein is in aliquid or semi-solid carrier suitable for intramuscular, intravenous,intramedullary or intra-articular injection.
 14. The method of claim 12wherein the bone morphogenic protein is selected from the groupconsisting of BMP-2, BMP-3, BMP4, BMP-6 and BMP-7.
 15. A method foraugmenting bone formation in an individual in need thereof whichcomprises administering to said individual thereof a bone formationaugmenting amount of the composition of claim
 2. 16. A method foraugmenting bone formation in an individual in need thereof whichcomprises administering to said individual thereof a bone formationaugmenting amount of the composition of claim 3.